This document provides information on various histochemical stains and markers used to identify structures in tissue samples under the microscope. It describes stains that can identify collagen, elastic tissue, nerve, fat, melanin, hemosiderin, calcium, mucin, amyloid, mast cells, bacteria, spirochetes, fungi, and epithelial, melanocytic, adnexal, muscle, neural, endothelial, and hematopoietic markers. Special stains and markers are essential tools for microscopic interpretation and diagnosis in dermatopathology.

1. Special stains and markers
Done by:
Zainab Nasser Almossalli
Junior dermatology resident

2. Histochemical
stains

3. The mixture
of hematoxylin and eosin (H&E) is the
routine staining choice for microscopic
Interpretation resulting in
blue nuclei and pink cytoplasm.

5. Collagen / muscle
 Masson trichrome :
Collagen: Blue or green
Muscle, nerves, nuclei: Dark red
very young collagen can stain red
 Verhoeff-van Gieson:
Collagen: Red
Nuclei, muscles, and nerves: Yellow
Aldehyde fuchsin (Gomori):
Collagen: Red

6. Mallory triple stain or trichrome stain (aniline blue):
Collagen: Blue
Muscle: Red
PTAH (phosphotungstic acid hematoxylin):
Collagen: Red
Muscle/fibrin: Blue
Movat’s pentachrome:
Collagen: Yellow
Muscle/fibrin: Red

8. Elastic tissue
Verhoeff-van Gieson:
Black
Orcein-Giemsa (Pinkus acid orcein) :
Black
Aldehyde fuchsin (Gomori):
Blue or purple
Movat’s pentachrome :
Black

9. Nerve
Bodian:
Black
Fat
Oil-red-O:
Requires fresh frozen tissue
Red
Sudan black B:Requires fresh tissue
Black
Scarlet red: Requires fresh tissue
Red, brown

10. Melanin
Fontana-Masson :
Black
Other uses: stain cryptococcus
Orcein-Giemsa:
Brown-black
Silver nitrate:
Black

12. Hemosiderin
Prussian blue (Perls stain):
• Hemosiderin/iron (Useful to distinguish melanin
from hemosiderin)
• Does not demonstrate iron in intact red blood
cells
Bright blue
Turnbull’s blue:
Iron/hemosiderin
Blue

14. Alizarin Red :
•Binds directly to calcium ions
• Reddish orange
Von Kossa:
•silver stain
• Brown black
Calcium

15. Alcian blue
• Acid MPS (pH 2.5) and Sulfated MPS (pH 0.5)
•Blue
•Alcian blue can be used with and without
hyaluronidase to differentiate hyaluronic acid
from other mucosaccharides.
Mucin/Mucopolysaccharides
In normal skin, most mucin is sulfated acid
mucosaccharide (heparin, chondroitin, and
dermatan sulfates).
In most pathologic states with increased
dermal mucin , the mucin is predominantly
nonsulfated hyaluronic acid

16. Aldehyde fuchsin (Gomori)
• Acid MPS, elastic tissue
• Blue
Colloidal iron
• acid MPS , Blue
Toluidine blue
• Acid MPS
• Purple (metachromatic)

18. Crystal violet:
• Acid MPS
•Purple with blue background
Mucicarmine:
• “Epithelial” mucin
•Bright pink ( Paget’s , cryptococcus
capsule)
Giemsa:
• Acid MPS
• Metachromatically purple

20. Periodic acid–Schiff (PAS):
• Stains glycogen, neutral mucopolysaccharides
(such as basement membrane), and fungi red.
• Glycogen ((clear cell acanthoma, trichilemmoma)).
• Fungi and neutral mucopolysaccharides basement membrane)
are diastase resistant, i.e., stain red with PAS after diastase
exposure.
(( tinea corporis, tinea versicolor, Candida, basement membrane
thickening of lupus erythematosus, thickened vessel walls
in porphyria)).

22. Congo red:
Pink -red, green double refractile with polarized light
Crystal violet:
Metachromatically stains Purple with blue background
Thioflavin T :
Yellow fluorescence under fluorescent Microscope
Scarlet red:
Red
Orcein-Giemsa: Light blue
Amyloid

24. Mast cells
Granules
Leder stain (chloroacetate esterase):
Red
Giemsa:
Purple
Toluidine blue:
metachromatically (the dye is blue but the granules
stain Purple)
Tryptase:
Red to brown

26. Bacteria
 Brown–Hopps ((modified gram stain)):
• A modification of the Brown–Brenn technique
Gram–positive organisms stain blue
Gram–negative organisms stain red
Giemsa:
• Giemsa stains many types of organisms,
including bacteria, Leishmania, and Histoplasma.
• Giemsa has many uses, including highlighting
myeloid and mast cell granules purplish blue.

27. Ziehl–Neelsen acid-fast stain
Fite acid-fast stain
Kinyoun’s acid-fast stain
• bright red
• Fite is preferred for “partially acid-fast” organisms such
As lepra bacilli, atypical mycobacteria, and Nocardia.
 Auramine-rhodamine:
• Requires a fluorescent microscope
• Mycobacteria fluoresce reddish-yellow
mycobacterium

29. Warthin–Starry (technically more
difficult than the others, “worthless Starry”)
Dieterle
Steiner (modified Dieterle Stain)
• Silver stains resulting in black spirochetes
• Examples: Lyme disease, syphilis
• Also stain Legionella, Bartonella, and Donovan
bodies of granuloma inguinale
Spirochetes

31. GMS (Gomori methenamine silver):
Donovan bodies, fungi
Black
PAS (Periodic acid-Schiff)
Fungi, neutral MPS, glycogen
Red
Grocott:
Fungi
Fungus cell wall: black
Fungi

32. special
Markers

33. Epithelial markers
Cytokeratin (Keratin):
 normal location: Epithelial cells, ± sweat glands
 positive in Epithelial tumors, some adnexal tumors
Types of keratin:
•AE1 (low molecular weight): basal epidermis,
sweat glands
•AE3 (high molecular weight): mid to superfi cial
epidermis
•CAM 5.2 : Paget’s disease; CK 7: Paget’s disease;
CK 20 : Merkel cell carcinoma

34. Ber-EP4:
 Marks most epithelial cells, but not those
undergoing squamous differentiation.
 Positive in basal cell carcinoma and negative
in squamous cell carcinoma.

36. S100
Normal location: Melanocytes, neural cells, smooth/skeletal
muscle cells, Langerhans cells, eccrine and apocrine glands,
chondrocytes.
Postive in: Langerhans cell histiocytosis, melanoma , granular cell
tumor, eccrine neoplasms, neural tumors, liposarcoma.
HMB-45 (less sensitive but more specific than S100):
Normal location:Melanocytes.
Postive in: Melanoma, some normal nevi, Spitz nevus,
angiomyolipoma, breast carcinoma.
Melanocytic

37. Melan A and Mart-1:
 Two different antibodies that stain the same
epitope.
Positive in: melanocytic lesions.
Do not stain desmoplastic melanoma reliably.

39. CEA (Carcinoembryonic antigen):
Normal location: Eccrine glands
Postive in: Paget’s disease, extramammary
Paget’s, epithelioid sarcoma (sometimes), sweat
gland tumors,adenocarcinomas (gastric, lung,
breast, etc.)
EMA (Epithelial membrane antigen):
Normal location: Eccrine glands, some epithelial
malignancies.
Postive in: Paget’s disease, merkel cell,
carcinoma meningioma, epithelioid sarcoma, most
epithelial tumors
Adnexal

41. Desmin:
Normal location: (skeletal and smooth muscle).
Positive in: Leiomyoma, leiomyosarcoma, glomus cell tumor
(focally +), embryonal rhabdomyosarcoma
Vimentin
Normal location: Muscle cells, fibroblasts, endothelial
cells, histiocytes, lymphocytes, Schwann cells, melanocytes.
Positive in: Mesenchymal tumors (sarcomas, lymphomas,
atypical fibroxanthoma, dermatofibrosarcoma protuberans,
melanoma, glomus cell tumor.
Smooth muscle actin:
Normal location: Myofibroblasts, muscle cells.
Positive in: Glomus tumors, smooth muscle tumors, some
atypical fibroxanthomas.
Muscle

43. Neuron-specific enolase (NSE):
Normal location: Nonspecific neuroendocrine marker.
Positive in: Granular cell tumor, merkel cell carcinoma,
other neuroendocrine tumors, some melanocytic tumors
(melanoma), schwannoma.
Glial fibrillary acidic protein (GFAP):
Normal location: Glial cells, astrocytes, Schwann cells.
Positive in: Nerve sheath tumors.
Neural

45. Chromogranin:
Normal location: Neuroendocrine neoplasms
Positive in: Merkel cell carcinoma
Synaptophysin:
Normal location: Neuroendocrine neoplasms
Positive in: Merkel cell carcinoma
Myelin basic protein(MBP):
Normal location: Myelin sheath tissue, Schwann cells
Positive in: Neurofibroma, neuroma, granular cell tumor

46. Factor VIII–relatedantigen ( von Willebrand):
Normal location: Endothelial cells(not as sensitive as
CD31)
Positive in: Vascular tumors (both benign and
malignant)
CD31: more specific in confirming vascular origin of
Tumors.
Normal location: Monocytes, granulocytes, T/B
cells,endothelial cells
Positive in : Vascular neoplasms (angiosarcoma)
Endothelial

47. Ulex europaeus agglutinin 1 (UEA):
Normal location: Endothelial cells, most eccrine glands,
keratinocytes
Positive in: Vascular tumors
CD34:
 normal location: Endothelial cells, nerves,
hematopoietic cells.
Positive in: Vascular tumors (benign/malignant),
dermatofibrosarcoma protuberans, vascular tumors
(not as sensitive as ulex), neurofibroma, epithelioid
sarcoma, spindle cell lipoma, fibrous papule.

49. Hematopoietic
CD45Ra (LCA):
 Leukocyte common antigen (LCA) is a general
marker of hematolymphoid differentiation
 present on all hematopoietic cells with the exception of
maturing erythroids and megakeratocytes.
CD45Rb:
 Loss of staining in epidermotropic T cells of mycosis
fungoides.
CD45Ro (UCHL-1): Mature T cells
CD20:
 B-cell antigen (often absent in plasma cells)
 Positive in B-cell lymphomas
 Target for rituximab.

50. CD79a: Plasma cell and B-cell marker
CD3: Pan-T-cell marker
 Positive in T-cell lymphomas
CD4: T-helper lymphocytic marker
CD8: T-cell cytotoxic/suppressor marker.
CD5: Pan-T-cell marker
 Positive in: mantle cell lymphoma and infiltrates of chronic
lymphocytic leukemia.
CD30 (Ki-1, BERH2):
Positive in: anaplastic large cell lymphoma ,lymphomatoid
papulosis, scabies nodules and chronic tick bites.

52. CD7: Immature T-lymphocyte antigen
 Most commonly lost antigen in T-cell lymphoma
CD56: Marker of NK cells and subsets of T cells
CD68 (KP-1): Reactive in monocyte/macrophage cells
Myeloperoxidase: granules of neutrophilic myeloid cells
 acute myeloid leukemia
ALK-1:
Anaplastic lymphoma kinase expressing chromosomal
translocation t(2,5)
 Positive in: most systemic anaplastic large cell lymphoma and
negative in primary cutaneous anaplastic large cell lymphoma.
Kappa/lambda:
Normally expressed in a ratio of two-thirds kappa to one-third
lambda
 10-fold deviation from this ratio suggests a clonal B-cell
proliferation

54. CD1a: Langerhans cells
CD43 (Leu-22): Pan-T-cell marker
CD20: is strongly suggestive of B-cell lymphoma
BCL2: oncogene that inhibits apoptosis
Positive in: nodal follicular center cell lymphomas,
Most basal cell carcinomas reveal diffuse staining,
whereas trichoepitheliomas only show staining of the
outermost epitheliallayers of the tumor islands

56. Proliferation marker
MIB-1 (Ki-67)
 More sensitive indicator of cell proliferation
than mitotic figures
 Helpful in lymphoma and melanoma

57. Factor XIIIa:
Normal location: Macrophages, dermal dendrocytes,
Platelets.
Positive in: Dermatofibroma, xanthoma disseminatum
,fibrous papule, Atypical FibroXanthoma , xanthogranuloma.
Bcl-2:
 Indicator of: ↓ apoptosis, ↑ cell longevity.
Positive in: Follicular center cell lymphoma.
Gross Cystic Disease Fluid Protein 15 (GCDFP-15):
Normal location: Apocrine glands.
Positive in: Apocrine tumors, breast carcinoma, salivary
gland tumors.
Miscellaneous

59. Quiz

60. 1. Biopsy of a lesion on the scrotum of
A 65 years old male shows pagetoid cells
In the epidermis.
Which of the following combination of
Studies may be helpful in diagnosing this
Case?
a.CK20,S100,Mart1,CK7 and CEA
b.CD1a,CD5,S100,PAS and GMS
c.CK20,CD1a,PAS,GMS
d.EMA,CD1a,CD5,CD3 and GMS

62. 2. A biopsy shows small lymphocytes in
Dense aggregates in the subcutaneous
Tissue. Immunohistochemical studies
Reveal that these are CD5 and CD20 positive.
The best diagnosis is:
A.AML
B.CML
C.ALL
D.CLL

63. 3. An immunocompromised patient present
With pulmonary lesion and wide spread
Papular eruption. Biopsy of a skin lesion
Revealed probable fungal organism that are
GMS and fontana masson positive.
The most likely diagnosis:
a. Coccidiomycosis
b. Cryptococcosis
c. Histoplasmosis
d. systemic candidiasis

64. 4. A 2year old boy has crusted ulcerated
Lesions on the scalp and forehead.
The drmatopathologist inform you that an
Immunohistochemical study shows numerous
CD1a positive cells in the epidermis.
What other study may be positive in this case?
a. S100
b. Mart1
c. CK7
d. CD5

65. 5. A 14 years old male has hypo pigmented
Patches on the elbow. You suspect vitiligo
And perform a biopsy.
Which of the following studies may be usful
In this case?
A. Fontana masson
B. PAS
C. Giemsa
D. Prussian blue

66. 6. A 23 year old HIV positive male present
With reddish lesions on his leg.
As a dermatologist what are going to
Write for the dermatopathologist after
tacking a biopsy?
A. Please perform a warthin starry stain
B. Please perform a leder stain
C. please perform an HHV_8 immuno study
D. please perform a CMV immuno study

67. References :
dermatopathology the requisites
dermatology illustrated
dermatology a pictorial review