Special stains are used in dermatopathology to enhance contrast in microscopic images and selectively stain cells, structures, and molecules. Common special stains include PAS for glycogen and basement membranes, Alcian blue for mucins, trichrome stains for collagen, Verhoeff-Van Gieson for collagen and muscle, Congo red for amyloid, Sudan stains for lipids, Giemsa and toluidine blue for mast cells, GMS and Fontana-Masson for fungi and melanin, Ziehl-Neelsen for acid-fast bacteria, and Gram staining for classifying bacteria. Each stain colors specific tissues or molecules to aid in diagnosis and examination of cellular components and structures.

1. SPECIAL STAINS
IN
DERMATOLOGY

2. WHAT IS STAIN?
 Staining is an auxiliary technique used in microscopy to enhance contrast in the microscopic
images.
 Stains may be used to define and examine the bulk tissue
1. Cell population
2. Muscle fibers
3. Connective tissues
4. Organelles

3. SPECIAL STAINS
 Used in addition to H & E staining to selectively
 Stain cells & cellular components
Gives information on:
 Presence of certain class of molecules
 Their localization
 Number of molecules

4. STAINS FOR
MUCOPOLYSACCARIDES
1. PAS
2. ALCIAN BLUE
3. MUCICARMINE
4. COLLOIDAL IRON

5. PAS
 Most commonly used special stain
 Stains neutral mucopolysaccharides (Glycogen)
 Basement membranes (e.g SLE)
 Fungi, Parasides
 Fibrin, Inclusion bodies

9. ALCIAN BLUE
 Best stain for mucin
 Alcian blue (pH 2.5)
- Acid mucopolysaccharides (glycosaminoglycain’s)
- Light blue
 Alcian blue (pH 0.5)
- Sulfated mucopolysaccharides (heparin sulfate)
- Blue
 Does NOT stain neutral mucins

10. RESULTS
 Acid mucin & proteoglycans : BLUE
 Nuclei : RED

12. MUCICARMINE
 To demonstrate acid & neutral mucopolysaccharides
- Epithelial mucin
- Capsule of cryptococcus neoformans
Results
 Acid epithelial mucins : Deep rose to red
 Nuclei : Black
 Others tissue elements : Light yellow

14. COLLOIDAL IRON
 A mucin stain for dermal/connective tissue mucin
 Stains acid mucopolysaccharides
RESULTS
 Proteoglycans , Acid mucins : BRIGHT BLUE
 Collagen : RED
 Muscle & cytoplasm : YELLOW

16. STAINS FOR PIGMENTS &
MINERALS
1. Fontana-Masson
2. Von-kossa
3. Alzarin red S
4. Prussian blue stain

17. FONTANA-MASSON
 Stains melanin & argentaffin granules – BLACK (nuclei will be red)
 Useful for quantifying melanocytes (e.g. in vitiligo) & in cases of minocycline pigmentary
alteration
 Also stains cryptococcus
RESULTS
 Melanin , Argentaffin, Chromaffin : BLACK
 Nuclei : RED

18. USES
 To identify melanin & argentaffin granules
 In diagnosis of malignant melanoma
 Argentaffin granules are found in carcinoid tumors

21. VON-KOSSA
 Stains calcium salts – BLACK
 Calcification of vessel walls & elastic tissue
( Calcinonis cutis, pseudoxanthoma elasticum, calciphylaxis, elastosis
& elastofibroma)

24. PRUSSIAN BLUE STAIN (PERLS IRON)
 Hemosiderin and ferric ions
 Useful for identifying iron as the source of pigment
 Stains iron – BLUE
RESULT
 Ferric iron : Blue
 Nuclei : Red

26. ALIZARIN RED
 Stain for calcium salts
RESULTS
 Calcium : ORANGE RED
 Background : GREEN

28. STAINS FOR CONNECTIVE
TISSUE
 Trichrome-Masson
 Verhoeff-Van Gieson

29. TRICHROME-MASSON
 Smooth muscle , cytoplasm, keratin – Red
 Useful for distinguishing leiomyoma’s from dermatofibromas & neural tumors
 Collagen – Blue/green
 Useful in evaluating the characteristics of dermal collagen
 Nucleus - Black

30. USES
 It is used to differentiate b/w collagen & smooth muscle in tumor
 To identified the increased collagen deposition : Keloid
RESULT
 Nuclei : Blue/Black
 Cytoplasm, Muscle, & Erythrocytes : Red
 Collagen : Blue

32. VERHOEFF-VAN GIESON OR WEIGERT
 Used to differentiate collagen and smooth muscle
 Can be used to demonstrate the presence of collagen in pathological conditions( DLE, EN, RA,
SLE, MORPHOEA)
 Stains nuclei : Blue
 Cytoplasm, muscle, fibrin : Yellow
 Collagen : Bright
 Elastic fibers : Black

34. STAINS FOR AMYLOID
 Congo red
 Thioflavin T
 Crystal violet

35. CONGO RED
 Stains amyloid : PINKISH-RED
 Gives apple-green birefringence to amyloid
 The most specific method for amyloid

38. THIOFLAVIN T
 Amyloid shows yellow fluorescence
RESULT
 UV light source : Silver-blue
 Blue light fluorescence : Yellow

40. CRYSTAL
VIOLET
• Stain Amyloid purple-violet

41. STAINS FOR FAT
 Sudan black B
 Sudan orange
 Oil red O

42. SUDAN BLACK B
 Most sensitive of all fat dyes
 Stains neutral fats – Blue-Black
 Stains phospholipid – Grey

44. OIL RED O
 Stains fat red
 Frozen/Fresh tissue ( once tissue is fixed &processed into paraffin blocks, this method does not
work)
 This may be very helpful in seeing the fat globules in sebaceous carcinoma

45. RESULT
Fat : Brilliant Red
Nuclei : Blue FAT
NUCLEI

46. STAINS FOR MAST CELLS
 Giemsa
 Toluidine blue
 Chloroacetate esterase ( Leder stain)

47. GIEMSA
Metachromatically purple
Urticaria
Mastocytosis MAST CELL

48. TOLUIDINE
BLUE
Mast cells stain – Purple
(Metachromatic staining)
Background stain – Blue
(orthochromatic staining)
Mastocytosis
MAST CELL

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

50. H&E
May demonstrate fungi, bacteria

51. GRAM STAINING
 Differentiates bacteria by the chemical & physical properties of their cell well by detecting
peptidoglycan
 Gram +ve bacteria retain the crystal violet dye & thus are stained violet : BLUE
 Gram –ve bacteria : PINK

53. GOMORI METHENAMINE SILVER (GMS)
 Fungal cell walls
 Stains fungi & parasites brown or black with a green background
 Pneumocystis carnii, histoplasma , leshmania

56. GIEMSA STAIN
 Used to stain bacteria & protozoa
 H. pylori, rickettsia & chlamydia
 Bacteria stains : BLUE
 Cytoplasm stains from : Pink to rose
 Nuclei : BLUE
 Eosinophils are also easily detected

58. WARTHIN-STARRY
 A silver nitrate stain
 Spirochetes – BLACK
 Background – Golden yellow

60. ZIEHL-NEELSEN
 Acid fast refers to cell walls containing high lipid content
 Used to stain Mycobacteria, oocysts of Cryptosporidium parvum, Cyclospora, Isospora
 Acid fast cells stain : RED
 Non acid fast cells stain : BLUE

62. ACID FAST STAINING
TECHNIQUES
 Ziehl-Neelsen stain ( classic type)
 Kinyoun
 Auramine-rhodamine (fluorescent dyes)
 Fite : for leprosy bacillus which is less acid fast

64. THANK YOU