H I S T O C H E M I C A L

S T A I N S
RJS GUCABAN-CALIMAG
 W H AT A R E

S P E C I A L S TA I N S
A N D W H Y A R E T H E Y I M P O RTA N T ?

• IMPORTANT TOOLS TO PATHOLOGISTS FOR THE VISUALIZATION AND IDENTIFICATION OF

SPECIFIC CELLS, TISSUES AND MICROORGANISMS
• BASED ON CHEMICAL REACTIONS THAT USED VARIOUS COLORED DYES AND REAGENTS
• TARGETED TO IDENTIFY:
⚬ SPECIFIC CELL TYPES (MUSCLE)
⚬ SPECIFIC TISSUE (COLLAGEN)
⚬ CELLULAR PRODUCTS (MUCIN)
⚬ SPECIFIC MORPHOLOGY (FUNGI)
 W H AT A R E

IMMUNOHISTOCHEMICAL
S TA I N S ( I H C )
A N D W H Y A R E T H E Y I M P O RTA N T ?

• USED TO DIFFERENTIATE TWO CONDITIONS WITH SIMILAR MORPHOLOGY, TO CONFIRM A

DIAGNOSIS, AS WELL AS TO ASSESS PROGNOSIS
• MORE COMMONLY USED FOR NEOPLASTIC CONDITIONS LIKE MELANOCYTIC,
HEMATOLYMPHOID, AND SPINDLE CELL TUMORS
• RESULTS SHOULD BE INTERPRETED IN TERMS OF
⚬ EXPRESSION (POSITIVE OR NEGATIVE),
⚬ PATTERN OF POSITIVITY (NUCLEAR, CYTOPLASMIC, OR MEMBRANOUS),
⚬ INTENSITY (WEAK OR STRONG), AND
⚬ EXTENT (FOCAL OR DIFFUSE).

CHATTERJEE, DEBAJYOTI; BHATTACHARJEE, RAJSMITA1. IMMUNOHISTOCHEMISTRY IN DERMATOPATHOLOGY AND ITS RELEVANCE IN CLINICAL PRACTICE. INDIAN
DERMATOLOGY ONLINE JOURNAL: JUL–AUG 2018 - VOLUME 9 - ISSUE 4 - P 234-244 DOI: 10.4103/IDOJ.IDOJ_8_18
HOW ARE SPECIAL STAINS
DIFFERENT FROM IHC?
• Special stains are based on dye chemistry that stains classes of cells, tissues or organisms (a broad
range, but limited specificity)
• IHC stains are based on antibody reactions with specific antigens that may be present on cell
surfaces, in the cytoplasm or in the nucleus (limited overall range but high specificity)
H I S T O C H E M I C A L S P E C I A L
S T A I N S

03
CONNECTIVE TISSUE STAINS
MAS S ON TR ICHR OME S TAIN

VERHOEF F -VAN GIES ON S TAIN

MASSON TRICHROME

• DIFFERENTIATES
COLLAGEN (BLUE-GREEN)
FROM SMOOTH MUSCLE
(RED)
• SCAR VS LEIOMYOMA
VERHOEFF-VAN GIESON

• ELASTIC FIBERS ARE BLACK

• absence or reduction
• distorted fibers
MAST CELL STAINS
TOLUIDINE B LUE

GIEMS A

LEDER S TAIN
TOLUIDINE BLUE

• STAINS MAST CELL

GRANULES
METACHROMATICALLY (I.E.,
THE DYE IS BLUE BUT THE
GRANULES STAIN PURPLE)
• Also stains mucin
GIEMSA

• STAINS MAST CELL

GRANULES
METACHROMATICALLY
• Also stains many types of
organisms and myeloid cells
LEDER (NAPHTHOL ASD CHLOROACETATE ESTERASE)

• MAST CELL CYTOPLASM

STAINS RED (NOT
DEPENDENT ON PRESENCE
OF GRANULES)
• Also stains myeloid cells
CARBOHYDRATE STAINS
P ERIODIC AC ID-S CHIF F

ALCIAN B LUE

COLLOIDAL IRON

TOLUIDINE B LUE

MUC ICAR MINE

PERIODIC ACID-SCHIFF
• STAINS GLYCOGEN,
NEUTRAL
MUCOPOLYSACCHARIDES
(SUCH AS BASEMENT
MEMBRANE), AND FUNGI
RED
• TINEA CORPORIS, TINEA
VERSICOLOR, CANDIDA,
BASEMENT MEMBRANE
THICKENING OF LUPUS
ERYTHEMATOSUS,
THICKENED VESSEL WALLS
IN PORPHYRIA

*Glycogen is diastase LABILE -clear cell

acanthoma, trichilemmoma
*Fungi and neutral mucopolysaccharides
(basement membrane) are diastase RESISTANT
ALCIAN BLUE

• DEMONSTRATES ACID
MUCOPOLYSACCHARIDES BY
STAINING THEM BLUE
• PATHOLOGIC STATES- MUCIN IS
NONSULFATED HYALURONIC ACID
⚬ FOLLICULAR MUCINOSIS
⚬ GRANULOMA ANNULARE
⚬ MYXOID CYST
⚬ DERMAL MUCIN IN LUPUS
ERYTHEMATOSUS
￭ ALL STAIN AT PH 2.5
COLLOIDAL IRON

• BLUE COLOR INDICATES ACID

MUCOPOLYSACCHARIDES
MUCICARMINE

• STAINS ACID
MUCOPOLYSACCHARIDES PINK TO
RED
• STAINS THE MUCINOUS CAPSULE OF
CRYPTOCOCCUS NEOFORMANS PINK
TO RED
AMYLOID
CONGO RED

THIOF LAVIN T

CRYS TAL VIOLET

CONGO RED
• AMYLOID STAINS BRICK RED
• DISPLAYS "APPLE GREEN"
BIREFRINGENCE WITH POLARIZED
LIGHT
CONGO RED

• AMYLOID STAINS BRICK RED

THIOFLAVIN T
• SECTIONS MUST BE EXAMINED
WITH A FLUORESCENT MICROSCOPE
• YELLOW TO YELLOW GREEN
APPEARANCE
CRYSTAL VIOLET

• METACHROMATICALLY RESULTS IN
A RED-PURPLE COLOR OF AMYLOID
IRON
P R US S IAN BLUE (P ERLS S TAIN)
PRUSSIAN BLUE (PERLS STAIN)
• FERRIC IRONS REACT TO FORM A
DEEP BLUE COLOR
• DISTINGUISHES MELANIN FROM
HEMOSIDERIN
• EX: PIGMENTED PURPURIC
DERMATOSIS
MELANIN
F ONTANA-MAS S ON
FONTANA-MASSON
• A SILVER STAIN THAT RESULTS IN A
BLACK PRECIPITATE WITH MELANIN
CALCIUM
VON KOS S A

ALIZAR IN RED
VON KOSSA
• SILVER STAIN THAT STAINS
CALCIUM SALTS BLACK
• PXE, CALCINOSIS CUTIS,
CALCIPHYLAXIS
LIPIDS
OIL RED O

S UDAN B LAC K

OS MIUM TETROXIDE
OIL RED O SUDAN BLACK
BACTERIA
BR OWN-HOP P S
BROWN-HOPPS

• A MODIFICATION OF THE BROWN–

BRENN TECHNIQUE
• GRAM-POSITIVE ORGANISMS STAIN
BLUE, AND GRAM-NEGATIVE
ORGANISMS STAIN RED
FUNGI
P ERIODIC AC ID S C HIF F

GROC OTT'S METHENAMINE S ILVER

PERIODIC ACID-SCHIFF

• FUNGI ARE PAS POSITIVE AND

DIASTASE RESISTANT
GROCOTT'S METHENAMINE SILVER

• GRAY-BLACK REACTION WITH

FUNGAL WALL
• Also stains Nocardia and Actinomyces
MYCOBACTERIA
ZIEHL–NEELS EN AC ID-FAS T S TAIN; FITE ACID-FAST
S TAIN; KINYOUN'S ACID-FAS T STAIN

AURAMINE-R HODAMINE
ZIEHL-NEELSEN, KINYOUN'S, FITE

• MYCOBACTERIA APPEAR BRIGHT RED

• FITE IS PREFERRED FOR “PARTIALLY
ACID-FAST” ORGANISMS SUCH AS
LEPRA BACILLI, ATYPICAL
MYCOBACTERIA, AND NOCARDIA
• FITE PRESERVES COLOR DUE TO USE
OF PEANUT OIL BEFORE STAINING
AND GENTLE DECOLORIZATION
AURAMINE-RHODAMINE

• REQUIRES A FLUORESCENT
MICROSCOPE
• Mycobacteria fluoresce reddish yellow
SPIROCHETES
WARTHIN-S TAR RY

S TEINER (MODIF IED DIETERLE STAIN)

WARTHIN-STARRY

• SILVER STAIN
• STAINS ORGANISMS DARK BROWN TO
BLACK, AND THE BACKGROUND
LIGHT GOLDEN BROWN/GOLDEN
YELLOW
STEINER

• SILVER STAINS RESULTING IN

BLACK SPIROCHETES
• EXAMPLES
⚬ Lyme disease (around vessels and in
dermal papillae)
⚬ syphilis (in lower epidermis)
• Also stains Legionella, Bartonella, and
Donovan bodies of granuloma inguinale
GIEMSA

• STAINS MANY TYPES OF ORGANISMS,

INCLUDING BACTERIA, LEISHMANIA, AND
HISTOPLASMA
I M M U N O H I S T O C H E M I C A L
S T A I N S

03
EPITHELIAL
MARKERS
AE1/AE3

• Cocktail of high- and low-

molecular-weight monoclonal
cytokeratin antibodies

• Expressed in the epidermis

and adnexal epithelium

• Stains all epithelial tumors

(squamous cell carcinoma and
adnexal tumors) and generally
excludes mesenchymal,
melanocytic, and
hematopoietic tumors

• Also stains epithelioid

sarcoma, synovial sarcoma
and mesothelioma
AE1/AE3
• AE1 detects the high molecular weight cytokeratins 10, 14, 15 and 16 and the low
molecular weight cytokeratin 19

• AE3 detects the high molecular weight cytokeratins 1, 2, 3, 4, 5 and 6 and the low
molecular weight cytokeratins 7 and 8

• Pankeratin, broad spectrum keratin or keratin: not preferred as this can also refer
to cytokeratin MNF116 and (to a lesser extent) CAM5.2
P63

• Expressed in basal and

spinous cells of the
epidermis, germinative cells
of sebaceous glands, and
myoepithelial cells of the
sweat glands

• Lack of reactivity in
metastatic carcinoma

• Identification of cutaneous
spindle cell CA from other
spindle cell neoplasm
CAM5.2

• Detects low-molecular-weight
cytokeratins present in most
glandular neoplasms without
staining the epidermis or
stratified squamous
epithelium

• Marks Paget disease and

extramammary Paget disease
CK7

• Used in determining the

origin of metastatic carcinoma

• In general, a marker of
adenocarcinomas that
originate above the
diaphragm (non-
gastrointestinal)

• Marks Paget disease and

extramammary Paget disease
CK7
CK20
• Marks Merkel cell carcinoma
predominantly in a
paranuclear pattern

• Used in determining the

origin of metastatic carcinoma

• In general, a marker of
adenocarcinomas that
originate below the
diaphragm (gastrointestinal)

• Sparse merkel cells of

desmoplastic
trichoepithelioma
THYROID TRANSCRIPTION FACTOR (TTF-1)

• Useful in the small blue cell

tumor differential diagnosis

• Reactive in metastatic small

cell lung carcinoma and
negative in Merkel cell
carcinoma
BER-EP4

• Marks most epithelial cells

but not those with squamous
differentiation

• (+) in BCC
EPITHELIAL MEMBRANE ANTIGEN (EMA)

• Highlights normal sebaceous

and sweat glands

• Positive in sebaceous
carcinoma, Paget and
extramammary Paget

• Positive in squamous cell

carcinoma but negative in
basal cell carcinoma
CARCINOEMBRYONIC ANTIGEN

• Sweat glands are

immunoreactive

• Positive in sweat gland

neoplasms, Paget,
extramammary Paget, and
most adenocarcinomas
ADIPOPHILIN

• Membrano-vesicular
expression in lipid droplets of
sebaceous and xanthomatous
lesions

• Can help distinguish

sebaceous carcinoma from
squamous cell carcinoma and
basal cell carcinoma
MESENCHYMAL
MARKERS
DESMIN

• Positive staining in skeletal

and smooth muscle, except
vascular smooth muscle

• Negative in myoepithelial
cells and focal or weak in
myofibroblasts
SMOOTH MUSCLE ACTIN

• Positive in smooth muscle,

including vascular smooth
muscle

• Also expressed in
myofibroblasts and
myoepithelial cells

• Negative in skeletal muscle

SMA DESMIN
CD34

• Marker of vascular
endothelium and
hematopoietic progenitor cells

• Positive in
dermatofibrosarcoma
protuberans and negative in
dermatofibroma

• Typically highlights the

stroma of trichoepitheliomas
but not basal cell carcinomas
FACTOR XIIIA

• Highlights a population of
dermal dendritic cells

• Positive in dermatofibroma
and negative in
dermatofibrosarcoma
protuberans

• Positive in fibrous papule of

the face
CD31

• Helpful in confirming
vascular origin of tumors

• More specific vascular

marker than CD34
D2-40

• Lymphatic endothelial marker

• Increases detection of
lymphovascular invasion

• Positive in cutaneous adnexal

neoplasms
GLUT-1 (GLUCOSE TRANSPORTER)

• Positive in infantile
hemangiomas (negative in
congenital hemangiomas
including RICH and NICH
and negative in vascular
malformations

• Also stains perineurial cells

and perineurioma
VIMENTIN

• Stains mesenchymal cells,

endothelial cells, fibroblasts,
melanocytes, lymphocytes,
and macrophages, but does
not react with keratinocytes
or other epithelium

• General marker of sarcomas

• Excludes most carcinomas

except rare spindle cell
carcinomas
NEUROECTODERMAL
MARKERS
S100

• Stains melanocytes,
Langerhans cells, sweat
glands, nerves, Schwann
cells, myoepithelial cells, fat,
muscle, and chondrocytes

• Useful in differential
diagnosis of spindle cell
neoplasms
⚬ desmoplastic melanoma
⚬ LCH
⚬ granular cell tumor
HMB-45

• Premelanosome marker

• Loss of staining of
melanocytes with descent into
the dermis is a manifestation
of loss of premelanosomes

• Loss of staining in deep

dermal component of most
benign nevi, but uniform
staining of blue nevi
MELAN-A AND MART-1

• Two different antibodies that

stain the same epitope

• Stain melanocytic lesions

• Do not stain desmoplastic

melanoma reliably
MICROPHTHALMIA-ASSOCIATED TRANSCRIPTION
FACTOR (MITF)

• Essential in development and

survival of melanocytes

• Nuclear melanocytic marker

• Positive in cellular
neurothekeomas
SOX-10

• Nuclear marker of Schwann

cells and melanocytes

• Sensitive marker of
melanoma, including
conventional, spindled, and
desmoplastic types
NEUROENDOCRINE
MARKERS
NEURON SPECIFIC ENOLASE (NSE)

• Positive in neuroendocrine
cells, neurons, and tumors
derived from them

• Positive in so many other cell

lines that it is sometimes
referred to as nonspecific
enolase
NEURON SPECIFIC ENOLASE, CHROMOGRANIN,
SYNAPTOPHYSIN IN MERKEL CELL CA
HEMATOPOIETIC
MARKERS
CD1A

• Stains Langerhans cells

• Examples: Langerhans cell

histiocytosis
CD3

• Pan-T-cell marker

• Positive in T-cell lymphomas

but negative in B-cell
lymphomas
CD4
• T-helper lymphocytic marker
CD7

• Immature T-lymphocyte
antigen

• Most commonly lost antigen

in T-cell lymphoma
CD10 (CALLA)
• Common acute lymphoblastic
leukemia antigen (CALLA) is
an early marker of B-cell
differentiation

• Useful in differential
diagnosis of B-cell
lymphoproliferative disorders

• Stains only the stroma of

trichoblastomas, the
epithelial cells of BCC

• Expressed in most atypical

fibroxanthomas
CD20
• B-cell antigen (often absent in
plasma cells)

• Positive in B-cell lymphomas

and negative in T-cell
lymphomas

• Target for rituximab. Loss

correlates with rituximab
resistance
CD30 (KI-1, BERH2)
• Originally identified on
Reed–Sternberg cells of
Hodgkin disease

• Positive in activated
lymphocytes of anaplastic
large cell lymphoma and
lymphomatoid papulosis
(large cell transformation)

• Many positive cells may be

seen in scabies nodules and
chronic tick bites
CD43 (LEU-22) CD45RA (LCA)
• Leukocyte common antigen
• Pan-T-cell marker
(LCA) is a general marker of
hematolymphoid
• Aberrant coexpression with
differentiation
B-cell marker CD20 is
strongly suggestive of B-cell
• Present on all hematopoietic
lymphoma
cells and their precursors with
the exception of maturing
erythroids and
CD45RO (UCHL-1) megakeratocytes
• Mature T cells
CD56
• Marker of NK cells and
subsets of T cells

• Stains blastic plasmacytoid

dendritic cell neoplasm
(formerly known as blastic
NK/T-cell lymphoma or
CD4+/CD56+ hematodermic
neoplasm)
CD68 (KP-1) CD79A
• Reactive in virtually all • Plasma cell and B-cell marker
monocyte/macrophage cells

CD138 (SYNDECAN-1) CD163

•Plasma cell marker •Reactive in monocytes and
macrophages
CD68 (KP-1)
CD117 (C-KIT)
• Expressed in mast cells and
melanocytes

• In nevi and primary

melanoma there is a decrease
in expression in the dermal
component

• Typically lost in metastatic

cutaneous melanoma
LANGERIN (CD207)
• Surrogate marker for presence
of Birbeck granules in
Langerhans cells
MYELOPEROXIDASE
• Major constituent of granules
of neutrophilic myeloid cells

• Marker for acute myeloid

leukemia
ALK-1
• Positive in most systemic
anaplastic large cell
lymphomas and negative in
primary cutaneous anaplastic
large cell lymphoma
• Those few patients with ALK-
1-negative systemic anaplastic
large cell lymphoma have a
poor prognosis
KAPPA/LAMBDA
• Normally expressed in a ratio
of two-thirds kappa to one-
third lambda
• Tenfold deviation from this
ratio suggests a clonal B-cell
proliferation
BCL2
• An oncogene that inhibits
apoptosis

• Useful in differential
diagnosis of B-cell
lymphoproliferative disorders

• Most basal cell carcinomas

reveal diffuse staining,
whereas trichoepitheliomas
only show staining of the
outermost epithelial layers of
the tumor islands
MULTIPLE MYELOMA ONCOGENE-1 (MUM-1)
• Expressed in plasma cells,
activated T cells, and subset
of germinal center cells

• Distinguishes primary
cutaneous diffuse large B-cell
lymphoma, leg type from
diffuse follicle center
lymphoma
PROLIFERATION
MARKERS
MIB-1(KI-67)
• Nuclear proliferation marker

• Not cell type specific

• Expressed in all active phases

of the cell cycle (G1, M, G2,
S)

• Pattern and number of

reactive melanocytes can be
helpful in diagnosing
melanocytic lesions
PHH3

• Mitotic marker that only

stains cells in the M phase of
the cell cycle

• Helps differentiate mitoses

from apoptotic or
hyperchromatic nuclei
 R EF ER E N C ES
Elston, Dirk, M. et al. Dermatopathology E-Book.
Available from: Elsevier eBooks+, (3rd Edition). Elsevier -
OHCE, 2018.

Chatterjee D, Bhattacharjee R. Immunohistochemistry in

Dermatopathology and its Relevance in Clinical Practice.
Indian Dermatol Online J. 2018 Jul-Aug;9(4):234-244. doi:
10.4103/idoj.IDOJ_8_18. PMID: 30050812; PMCID:
PMC6042184.
CASES
7
CASE 1
 A. WHAT IS YOUR DIAGNOSIS?
B. STAINS TO REQUEST TO AID IN
THE DIAGNOSIS?
A. CUTANEOUS MASTOCYTOSIS
B. GIEMSA, TOLUIDINE BLUE,
LEDER STAINS
CASE 2
 AT THIS POINT, WOULD YOU
WANT TO REQUEST FOR STAINS
TO HELP IN YOUR DIAGNOSIS?
A. WHAT IS YOUR DIAGNOSIS?
A. MYCOSIS FUNGOIDES
CASE 3
 AT THIS POINT, WOULD YOU
WANT TO REQUEST FOR STAINS
TO HELP IN YOUR DIAGNOSIS?
CD34 FACTOR XIIIA
WHAT IS YOUR DIAGNOSIS?
DERMATOFIBROSARCOMA
PROTUBERANS
CASE 4
 AT THIS POINT, WOULD YOU
WANT TO REQUEST FOR STAINS
TO HELP IN YOUR DIAGNOSIS?
CD10
DIAGNOSIS?
 ADDITIONAL STAINS:
• BER-EP4- DIFFUSE
POSITIVITY OF TUMOR CELLS
BASAL CELL CARCINOMA
THANK YOU!