stains in dermatology

1. STAINS IN DERMATOLOGY
Done by : Dr.Narjes Abdalqader
Supervised by : Dr.Ameen Alma’yta

2.  Hematoxylin and eosin stain (H&E)
 Special stains
 Immunofluoresent stain
 Immunohistochemical stains

3. H&E STAIN
 The standard stain in dermatopathology
 Hematoxylin…basic dye..
 basophilic structures: cellular nuclei and the granular layer
of the epidermis
 Blue
 Eosin .. Acidic dye ..
 eosinophilic structures: cytoplasm,collagen, muscle, nerve
and fibrin
 Pink - red

5. SPECIAL STAINS

6. H&E SPECIAL STAINS

8. SPECIAL STAINS
 Mucopolysaccarides
 Minerals AND pigments
 Connective tissue
 Fat
 Amyloid
 Microorganisms

9. STAINS FOR MUCOPOLYSACCARIDES
 PAS
 alcian blue
 Mucicarmine
 Colloidal iron

10. o Most commonly used special stain
o Stains Glycogen, fungal walls, neutral mucopolysaccharides,
fibrin, basement membranes
 thickened epidermal basement membrane in lupus
erythematosus and the thickened vascular walls in porphyria
cutanea tarda
 RED
PAS STAIN

11. SLE

12. Candida

13.  Best stain for mucin
 Alcian blue (pH 2.5):
 Acid mucopolysaccharides (glycosaminoglycans)
 Light blue
 Alcian blue (pH 0.5) :
 Sulfated mucopolysaccharides (heparinsulfate, chondroitin
sulfate)
 Blue
 does NOT stain neutral mucins
Alcain blue

14. MUCIN DEPOSITION

15. ALCIAN BLUE AND PAS STAINING PROTOCOL
 Demonstrate presence of mucin
 acidic mucins …… Alcian blue
 neutral mucins …….PAS
 Diastase pre-treatment may be used to eliminate the
presence of glycogen, prior to staining in Alcian blue.

16. Mucicarmine
oEpithelial mucin
o(acid or neutral mucopolysaccharides)
oUsually used for :
osialomucin (e.g. adenocarcinoma, Paget’sdisease)
the capsule of Cryptococcus neoformans
Red

17. Cryptococcus neoformans

18. COLLOIDAL IRON
 Acid mucopolysaccharides
 Blue
 Mucinoses
 Lupus erythematosus
 Extramammary Paget’s disease

20. STAINS FOR PIGMENTS AND MINERALS
 Fontana-Masson
 Von-Kossa
 Alzarin red S
 Prussian blue stain

21. FONTANA-MASSON
 Melanin
 Black
 Distinction between iron and melanin
 Discoloration due to drugs (e.g. minocycline)
 Evaluation of vitiligo

22. Enhancement of increased melanin within the basal layer of the
epidermis in genital melanosis

24. VITILLIGO WITH DEPEGMENTED AREAS

25. VON-KOSSA
 Calcium salts
 treated with a silver nitrate solution and the silver is deposited
by replacing the calcium reduced by the strong light, and
thereby visualized as metallic silver.
 Black
 Pseudoxanthoma elasticum
 (oxalate salts may not stain with this method)

27. ALIZARIN RED
Stains calcium red

28. • PRUSSIAN BLUE STAIN(PERLS’ IRON )
 Hemosiderin and Ferric ions
 Useful for identifying iron as the source of pigment
 Stains iron blue

29. demonstration of iron (hemosiderin) within
dermal macrophages

31. STAINS FOR CONNECTIVE TISSUE
 Trichrome-Masson
 Verhoeff-van Gieson

32.  Smooth muscle , cytoplasm , keratin
 red
 Useful for distinguishing leiomyomas from dermatofibromas
and neural tumors
 Collagen
 Blue / green
 Useful in evaluating the characteristics of dermal collagen
 Nucleus
 black
Trichrome-Masson

34. VERHOEFF–VAN GIESON OR WEIGERT
 used to differentiate between collagen and smooth muscle in
tumours
 AND to demonstrate the increase of collagen in diseases.
 Collagen
 Pink to red
 Elastic tissue
 Black
 Muscle and nerves
 Yellow
 Elastic tissue disorders (e.g. PXE, anetoderma, mid-dermal
elastolysis)

35. LOSS OF ELASTIC FIBERS IN THE SUPERFICIAL
DERMIS.(ANETODERMA(

36. STAINS FOR AMYLOID
 In H&E stain , can be confused with hyaline and fibrinoid
substances
 Congo red
 Thioflavin T
 Crystal violet

37. CONGO RED
The most specific method for amyloid
 Stains amyloid red
 green in polarized light

38. THIOFLAVIN T
 • Amyloid shows yellow fluorescence

39. • CRYSTAL VIOLET
 Stains Amyloid purple-violet

40. STAINS FOR FAT
 Sudan black B
 Sudan orange
 Oil red O

41. Most sensitive of all fat dyes
Sudans must be dissolved in organic solvents to
penetrate fats
stains neutral fats blue -black
 stains phospholipids gray
Sudan black B

42.  It fails to stain crystalline cholesterol, lecithin and free fatty
acids
 Bromine pre treatment converts crystalline cholesterol to
oily derivatives permeable to Sudan dye

43. OIL RED O
 Stains fat red
 frozen/fresh tissue (once
tissue is fixed and processed
into paraffin blocks, this
method does not work).
 This may be very helpful in
seeing the fat globules in
sebaceous carcinoma

45. STAINS FOR MAST CELLS
 Giemsa
 toluidine blue
 chloroacetate esterase (Leder stain)

46. GIEMSA
 Metachromatically purple
 Urticaria
 Mastocytosis
mastocytosis

47. TOLUIDINE BLUE
 Mast cells stain purple
 (Metachromatic staining)
 the background stain blue
 (orthochromatic staining)
 Mastocytosis

48. MASTOCYTOSIS

49. CHLOROACETATE ESTERASE (LEDER STAIN)
 Myeloid cells and mast cells

 Red
 Neutrophilic dermatoses
 Malignant hematopoietic infiltrates
 Mastocytosis

51. STAINS FOR MICROORGANISMS
 H&E
 Gram
 Giemsa
 Gomori methenamine silver (GMS)
 PAS
 Fontana-Masson
 Warthin-Starry
 Ziehl-Neelson stain

52. H&E
 May demonstrate fungi, bacteria

53. GRAM STAINING :
 differentiates bacteria by the chemical and physical
properties of their cell wall by detecting peptidoglycan ,
which is present in the cell wall of Gram-positive
bacteria.
 Gram-positive bacteria retain the crystal violate dye and
thus are stained violet - blue
 the Gram-negative bacteria do not; after washing, a
counterstain is added (commonly safranin or fuchsine)
that will stain these Gram-negative bacteria a pink color.


55. GOMORI METHENAMINE SILVER (GMS)
 Fungal cell walls
 Stains fungi and parasites brown or black with a
green background
 Pneumocystis carnii, histoplasma spp, leshmania

57. Cryptococcus

58. GIEMSA STAIN :
 Nuclei of cells, microorganisms (bacteria and
protozoa, H. pylori, rickettsia and chlamydiae)
 Blue
 Leishmaniasis
 Histoplasmosis
 Granuloma inguinale
 Mast cell granules
 Metachromatically purple
 Urticaria
 Mastocytosis

59. GRANULOMA INGUINALE

60. WARTHIN-STARRY
 A silver nitrate stain
 SPIROCHETES – black
 BACKGROUND – golden -yellow

61. Treponema pallidum

62. ZIEHL–NEELSEN
 Acid-fast :
 cell walls contain a waxy substance composed of
mycolic acids that present a barrier to dye entry as
well as elusion (washing out with solvent)
 Can be stained by a strong stain like carbol fuchsin
 Acid fast cells stain Red

63. ZIEHL-NEELSEN

64. ACID FAST STAINING TECHNIQUES
 Ziehl–Neelsen stain (Classic type)
 Kinyoun
 Auramine-rhodamine ( fluorescent dyes)
 Fite: for leprosy bacillus which is less acid fast ….
magenta

66. IMMUNOFLUORESCENCE METHODS
 A technique for detecting the presence and position
of antigens, antibodies, other cell components.
 The principle of the technique :
 certain fluorochrome dyes( flourecein and
rhodamine ) when exposed to ultraviolet (UV) light
emit fluorescent radiation with certain colors (green
and orange)

68. direct immunofluorescense :
 Pt own skin or mucous membrane
 Looking for Ag
 Bullous disease
 Dermatitis herpitiformis
 Lichen planus
 Lupus erythematosus
 Vasculitis (e.g. Henoch-Schonlein purpura)
 Porphyria


69. INDIRECT IMMUNOFLUORESCENCE :
 Looking for Ab
 Pt serum
 Substrate :
 Normal skin
 monkey esophagus : desg 1+3 …. pv
 murine bladder: plakin rich ….paraneoplastic
pemphigus

70. SITE OR THE BIOPSY :
 Bullous disease :
 From unblistered perilesional area
 Dermatitis herpitiformis
 Perilesional area
 Vasculitis :
 Very fresh lesion
 Lichen planus :
 Inflammed mucosa or skin
****Quick frozen or placed in michel transport media for
later quick freezing

72. BULLOUS PEMPHIGOID
LINEAR DEPOSITION ALONG EPIDERMAL BM

73. IIF ,,, BP,, N-SERRATED
CIRCULATING IGG AUTOANTIBODIES BIND TO THE
EPIDERMAL SIDE OF THE SALT-INDUCED SPLIT ;

74. IIF ,,EBA ,,, U-SERRATED
( WITH IGG ANTIBODIES AGAINST LAMININ 5/332)
REACT WITH THE DERMAL SIDE

75. IMMUNOHISTOCHEMISTRY (IHC)
 is the use of immunologic techniques to identify
cellular antigens (proteins) not visible on routine
H&E-stained sections.
 antibody is conjugated to an enzyme that can
catalyze a color-producing reaction when the
antibody–enzyme conjugate is bound to the
appropriate antigen within tissue
 the enzyme is often peroxidase ,hence the older
terminology, immunoperoxidase technique

76. IMPORTANT FACTORS TO CONSIDER WHEN
USING IHC
 (1) practically no antibody is specific for a certain cell type,
and therefore a panel of antibodies should be employed to
avoid premature, incomplete or erroneous conclusions;
 (2) a differential diagnosis must be constructed prior to
ordering an antibody panel so that the requested
antibodies are appropriate;
 (3) no antibody can differentiate irrefutably between a
benign and malignant neoplasm

77. Dermal infiltrate comprised of
lymphocytes and plasma cells (h&e)
antibody that recognizes a spirochetal
antigen
(immunoperoxidase technique)
syphilis

78. melanoma

79. EPITHELIAL MARKERS
 Bcl-2
 Protein product of an oncogene that negatively regulates
programmed cell death (apoptosis)
 Expressed in the basal layer of the epidermis and in
lymphoid cells
 distinguishing basal cell carcinoma from trichoepithelioma
 suggesting a systemic form of B-cell lymphoma when there is
cutaneous involvement
 CEA (carcinoembryonic antigen)
 A protein expressed by a variety of tissues, from
gastrointestinal cells to skin adnexa
 Expressed in eccrine and apocrine epithelium
 Paget’s disease (mammary and extramammary

80. MELANOCYTIC AND NEURAL MARKERS
 S100
 Family of low-molecular-weight, calcium-binding
proteins
 Expressed in neural crest-derived cells (melanocytes,
Schwann cells, glial cells), chondrocytes, fat cells,
macrophages, Langerhans cells, dendritic cells, some
breast epithelial cells and sweat glands
 Most sensitive marker for melanoma and spindle cell/
desmoplastic melanoma
 Malignant peripheral nerve sheath tumors (stains
weakly incomparison to melanoma)
 Clear cell sarcoma

81. MESENCHYMAL CELL MARKERS
 Desmin
 Intermediate filament protein expressed
predominantly by striated and smooth muscle cells
 Smooth and striated muscle cell neoplasms

82. MAST CELL MARKERS
 CD117 (c-KIT)
 Stem cell factor (KIT ligand) receptor
 Tyrosine kinase receptor expressed on the surface of hematopoietic
stem cells, myeloid progenitor cells, mast cells and melanocytes
 Mastocytosis, mast cell neoplasms
 Myeloid leukemia
 Junctional component in melanocytic nevi, some melanomas
 Gastrointestinal stromal tumors (GIST)
 The efficacy of imatinib, a c-KIT inhibitor, is affected by the
mutation status of the gene that encodes this receptor
 Tryptase
 Serine protease in mast cell granules
 Mastocytosis, mast cell neoplasms

83. MICROORGANISMS
 Cytomegalovirus (CMV)
 Glycoproteins expressed during the immediate-early and early
stages of CMV replication within infected cells
 CMV infection
 Epstein-Barr virus (EBV)
 EBV membrane protein encoded by BNLF
 Post-transplantation lymphoproliferative disorders, Hodgkin
disease
 Herpes simplex virus (HSV)
 Glycoproteins present within the viral envelope and core
 HSV-1 or -2 infection
 Human herpesvirus-8 (HHV-8)
 HHV-8 latency-associated nuclear antigen
 Kaposi sarcoma, primary effusion lymphoma, Castleman’s
disease

84.  Varicella-zoster virus (VZV)
 Glycoprotein I of VZV
 Varicella or herpes zoster
 BCG
 Anti-Bacillus Calmette-Guerin
 Mycobacterial infections
 Mycotic infections
 Bacterial infections
 Borrelia burgdorferi
 Reacts against various antigens (e.g. flagellin) of Borrelia burgdorferi
 Cross-reacts against Treponema pallidum, B. hermsii, and B. parkeri
 Lyme disease
 Syphilis
 Treponema pallidum
 Specific for all Treponema pallidum antigens
 Syphilis