Stains

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This article deals with stains. H&E isn't the only stain out there.

Non-H&E stains are often referred to as special stains.

Contents

Where to start...

Principles

When considering additional (i.e. special) stains one should (in order) do the following:[1]

  1. Make sure one has exhausted the clinical history; history is considered the best special stain.
  2. Special stains (below).
  3. Immunohistochemistry (dealt with in a separate article).
  4. Molecular testing, electron microscopy.

Common stains

  1. H&E stain.
  2. PAS stain.
  3. PAS-D stain.
  4. AFB stains, e.g. Ziehl-Neelsen stain.
  5. Congo red.
  6. GMS stain.
  7. Gram stain.

Immunohistochemistry

General

  • Abbreviated IHC.

Interpretation

Simple version:

  • Positive is (usually): brown.
  • Negative tissue is: light blue.

Important notes:

  • One has to know where the target (of the antibody) is supposed to be, i.e. cytoplasm vs. cell membrane.
  • The edge of the tissue may have light staining - edge effect.
  • If everything is brown... suspect that it didn't work.
  • In some situations you're blessed with an internal control, e.g. in renal tumours CD10 will stain RCC and the proximal tubule, in GISTs - CD117 the mast cells are positive.

Work-up of infection

It often not possible to be definitive by staining.[2]

Basic panel:

  • Gram stain - for bacteria.
  • GMS stain - fungal stain.
  • PAS (or PAS-D) - fungal stain.

Fungi

Fungi are a type of microorganisms. They are seen by pathologist every once in a while.

Specific stains

What follows is a big list... of stains.

Haematoxylin and eosin stain

  • Abbreviated H&E.

Haematoxylin phyloxin saffron stain

General

  • Abbreviated HPS.
  • An alternative to the H&E stain - some pathol. departments use this as their standard.

Interpretation

  • Haematoxylin = blue -- stains nucleus.
  • Phyloxin = pink -- stains muscle and cytoplasm.
  • Saffron = yellow -- stains collagen.
  • An alternative to H&E stain.
    • Fibrosis is easier to see on HPS than H&E... as one can see the collagen.

Images

Periodic acid Schiff stain

  • Abbreviated PAS.

Primary application

  • Kidney biopsies, medical.
  • Liver biopsies, medical.
    • Positive in alpha-1 antitrypsin deficiency.

Utility

  • Stains - lipofuscin,[3] basement membranes, fungi, glycogen, (neutral) mucin.

Interpretation

  • Magenta = glycogen, mucin, fungi.
  • Blue = nuclei.

Ref.:[4]

Image

Periodic acid Schiff fungal stain

  • Abbreviated PASF.

Primary application

Interpretation

  • Light purple = fungi.
  • Light green = background.
  • Washed-out light purple = Gram positive bacilli.

Note:

  • This is much improved over the PAS in the context of skin, as the background is similar to the fungal organisms.

Periodic acid Schiff with diastase

  • Abbreviated: PAS-D and PASD.

General

  • Diastase digests the glycogen.
  • "PAS diastase resistant"[5] implies PASD +ve and PAS +ve.

Use

  • Stains mucin.
  • Used to identify glycogen (together with PAS stain).
    • Glycogen = clear (digested) on PAS-D.
    • Glycogen = magenta on PAS.

Notes: [6]

Interpretation

  • Light purple = fungi.
  • Light blue/pink = background. ???

Gomori methenamine-silver stain

  • Abbreviated GMS.

Note:

  • GMS is "Grocott's methenamine Silver" according to WMSP.[7]

Use

Image

Acid-fast bacilli stains

  • Abbreviated: AFB.

There are several AFB stains:

Ziehl-Neelsen stain

  • Most popular acid-fast bacilli stain.
  • Stains other mycobacteria -- not specific for tuberculosis.
    • Stains Nocardia.[9]

Image

Fite stain

Interpretation:

  • Red = AFB.
  • Blue = background.

Auramine-rhodamine stain

  • Fluorescent stain.

Image

Kinyoun stain

Congo red stain

Use

  • Used to look for amyloid.
    • Mnemonic: CRAP = congo red amyloid protein.
  • An alternate stain for amyloid is Thioflavin T.

Note:

  • Thick sections (~10 micrometers) are considered a requirement for the stain to work properly.[12]
    • If the section is too thin... it doesn't work.

Interpretation

  • Amyloid = pink/red.
  • Nuclei = blue.

Ref.:[13]

Image

Thioflavin T stain

Use

Interpretation

  • Amyloid = green.

Image: Amyloid (inano.au.dk).

Gram stain

Use

  • "It is useless for finding bacteria."[14]
    • If they are to be seen... they'll be visible on H&E.

Note:

  • Microbiology is better at finding organisms than pathology.
    • They have one significant advantage -- if a small amount of bugs are present... they grows into a large (obviously visible) colony.

DDx for common patterns

A short list of bacteria and their characteristics:[15]

Shape\Gram stain Positive Negative Variable or negative
Bacilli Clostridium difficile, Bacillus anthracis, Nocardia spp. Escherichia coli, Helicobacter pylori, Yersinia pestis, Hemophilus influenzae Mycobacterium tuberulosis, Legionella pneumophila[16]
Cocci Streptococcus pneumoniae, Staphylococcus aureus Neisseria meningitidis, Moraxella catarrhalis

Interpretation

  • Purple (or blue) = Gram positive organisms.
  • Red = Gram negative organisms, nuclei.[17]
  • Yellow = background.

Notes:

  • Many of the bacteria are quite small relative to lymphocytes; Escherichia coli is 1-2 micrometers long x 0.25 micrometers in diameter.[18]
  • Epithelial cell nuclei & stromal cell nuclei may stain red.
  • Memory device: purple = positive.

Images

Luxol fast blue stain

  • Abbreviated LFB.

Use

  • Neuropathology, myelin stain.

Intepretation

  • Blue = myelinated fibers (contain lipoproteins), lipofuscin.[19]
    • Lack of blue (where it ought to be) = demyelination.
  • Purple = nerve cell (e.g. neuron).
  • Neutrophils = pink.

Ref.:[20]

Image

Giemsa stain

Use

Interpretation

  • Tissue is light blue/green.
  • Goblet cells are purple.[22]

Image:

Reticulin stain

Use

  • Liver biopsy, medical.
    • Demonstrates the reticular fibers (in cirrhosis the fibers are disrupted).
  • Before IHC, reticulin was used to differentiate sarcomas from carcinomas:[24]
    • Sarcomas have reticulin around each cell.
    • Carcinomas have reticulin around clusters of cells.
  • Commonly used in neuropathology.
    • In adenoma, reticulin highlights the lost acinar structure of normal pituitary gland.
    • Paraganglioma (Zellballen architecture)
    • Separating schwannoma (basement membrane around each cell) from meingioma in cerebellopontine angle.
    • Separating desmoplastic medulloblastoma from classic/anaplastic forms.


Interpretation

  • Black = reticular fibers.
  • Red = nuclei.

Notes:[25]

Images

Cresyl violet stain

Use

  • Used at some places (e.g. SMH) to look for Helicobacter organisms.

Interpretation

  • Everything is shades of blue.
    • Helicobacter stains blue.

Prussian blue stain

  • AKA Perl's iron stain.

Use

  • Useful for iron and hemosiderin; useful for differentiating brown pigments (melanin, lipofuscin, tattoo pigment, hemosiderin).

Interpretation

  • Blue = iron.

Image:

Notes:

  • Described well by vetmed.vt.edu.[26]
  • DDx of brown pigment: Fontana-Masson (melanin), Kluver-Barrera stain (lipofuscin).

Images

Kluver-Barrera stain

Combination of:

  • Luxol Fast Blue,
  • Cresyl Violet,
  • Special component for lipofuscin.

Use

  • Useful for differentiating brown pigments (melanin, lipofuscin, tattoo pigment, hemosiderin).
    • Stains lipofuscin.
  • Useful to detect demyelinating lesions in the CNS.

Notes:

  • PAS also stains lipofuscin and is more commonly available.

Interpretation

  • Blue pigmented granules = lipofuscin.

Notes:

  • Described well by vetmed.vt.edu.[27]
  • DDx of brown pigment: Fontana-Masson (melanin), Prussian blue stain (hemosiderin).

Oil red O stain

Use

  • Stains adipose tissue.
  • Corroborate diagnosis of lipoid pneumonia.[28]
  • Screen for GERD - positive staining seen in macrophages from BAL specimens.[29]
  • Uncommon.

Notes:

  • Must be done on fresh tissue, i.e. it cannot be fixed in formalin.

Interpretation

  • Red = fat.

Images

Warthin-Starry stain

Background:

  • Developed by a bunch of pathologists in Michigan to look for spirochetes.[30]

Use

  • Find spirochetes, e.g. syphilis (Treponema pallidum),[31] cat-scratch disease (Bartonella henselae).
  • Find Helicobacter spp., e.g. Helicobacter pylori -- Mount Sinai Hospital.[32]

Interpretation:[33]

  • Spirochetes - black.
  • Background - yellow.

Image

Notes:

  • Considered a "dirty" stain - picks-up junk in the background.[34]

Dieterle stain

Considered a variant of the Steiner stain.[35]

Use

  • Find spirochetes, e.g. syphilis (Treponema pallidum),[36] donovan bodies (leishmaniasis),[37] Helicobacter pylori and Bartonella henselae (Cat-scratch disease).[38]

Interpretation

  • Spirochetes - black.
  • Background - yellow.

Images

www:

Bielschowsky stain

Abbreviated: Biel stain.

Use

  • Stains glial tissue, i.e. brain.
    • Demonstrates neurofibrillary tangles, senile plaques (as in Alzheimer's disease).

Interpretation

  • Black = axons, tangles, plaques.
  • Brown/dark brown = plaque, vascular amyloid.
  • Yellow/brown = other.

Ref.: [39]

Image

Mucicarmine stain

  • Stains some mucins... uses the dye carmine.

Use

  • Identify mucin.
  • Malignant cells that produce mucin... carcinomas.[40]

Interpretation

  • Carmine with metanil yellow and Weigert's Hematoxylin:[41]
    • Blue/black = nucleus.
    • Yellow = background.
    • Red = mucin.[42]

Images

www:

Alcian blue stain

General

  • Stains acidic mucin (pH=2.5); Alcian blue = Acidic.
    • A variant uses pH=1.0.[7]

Note:

  • Alcian blue (not otherwise specified) usu. refers to the pH=2.5.[43]

Use

Note:

  • Esophageal submucosal glands - alcian blue positive.

Interpretation

  • Blue = acidic mucins.[44]

Notes:

Image

Sodium sulphate-alcian blue stain

Sulfated alcian blue (abbreviated 'SAB) redirects here.

Use

Interpretation

  • Green = amyloid.[45]
    • Other things that are green: mast cells, mucoid degeneration, basophilic myofibre degeneration, califications.
  • Yellow = background.

Image:

Movat's stain

Use

Components

  • Acid fuchsin, alcian blue, crocein scarlet, elastic hematoxylin, and saffron.[47][48]

Interpretation of Movat stain

  • Black = nuclei and elastic fibers.
  • Yellow = collagen and reticular fibers.
  • Blue = mucin, ground substance.
  • Red (intense) = fibrin.
  • Red = muscle.

Reference: [48]

How to remember? A.: Primary colours (red, blue, yellow) + black.

Images

Masson's trichrome stain

General

  • Collagen vs. muscle.

Interpretation

  • Black = nuclei.
  • Red = muscle (smooth muscle actin).
  • Baby blue = collagen.

Notes: [49]

Elastic trichrome stain

General:

  • "Elastic trichrome" is one important variant of Masson's trichrome.

Interpretation - as above in Masson's trichrome - plus:

  • Black = nuclei and elastin.

Mallory trichome stain

General

  • Collagen vs. muscle.
  • May be done with elastin.

Site

  • Kidney Bx (to assess for fibrosis).
  • Liver Bx (to assess for cirrhosis).
  • Cardiovascular/lung (to see differentiate the layers of the arteries, and arteries from veins).

Interpretation

  • Black = nuclei.
  • Red = muscle (smooth muscle actin).
  • Green = collagen.

Image

Haematoxylin orcein phyloxin saffron stain

  • Abbreviated HOPS.[50]
  • It should not be confused with the HPS stain.

Interpretation

  • Blue (haematoxylin) = nuclei.
  • Black (orcein) = elastin.
  • Red (phyloxin) = muscle.
  • Yellow (saffron) = collagen.

Jones stain

  • AKA PAS methenamine technique.[51]
  • AKA Methenamine PAS, abbreviated MPAS.

Use

  • Visualize basement membrane in kidney biopsies.

Interpretation

  • Black = basement membrane.
  • Blue = nuclei.
  • Pink = other structures/background.

Notes:[52]

Images

Hale's colloidal iron stain

Use

Notes:

Interpretation

  • Blue (granular cytoplasmic) staining is positive.[7]

Images:

Notes:

  • Often described as a "fastidious" (difficult/demanding) stain.[54]
    • A few staff think this is a totally useless stain.[55]
  • A variant exists known as the Muller and Mowry modification of Hale's colloidal iron stain (AKA Müller-Mowry stain).[56]

von Kossa stain

Use

  • Look for calcium.

Interpretation

  • Black = calcium.[7]

Toluidine blue stain

Use

Interpretation

  • Dark blue - nuclei, mast cell granules (darker than nuclei).
  • Light blue - cytoplasm.
  • Red/magneta - cartilage. (???)

Refs: looks a bit sketchy[59], [60]

Image

www:

Romanowsky stain

  • Occasionally spelled Romanowski.
  • Many variants of this stain exist.
  • Specimens are air-dried.

Interpretation:[61]

  • Red - RBCs, eosinophil granules.
  • Blue (basophilic) - lymphocyte cytoplasm.
  • Purple - nuclear chromatin, neutrophil granules, platelets.

Field stain

  • Variant of the Romanowsky stain for rapid processing.
  • Tends to "blow-up" cell, i.e. cells are larger vis-a-vis Pap stain.

Diff-Quik

Pronounced Diff-Quick.
  • Proprietary variant of Romanowsky stain.[62]

Uses:

Images

Wright stain

  • A variant of the Romanowsky stain; popular in North American.

Use:

  • Blood films.

May-Grünwald-Giemsa stain

  • A variant of the Romanowsky stain; popular in Europe.
  • Abbreviated MGG.

Use:

  • Blood films.
  • Cytopathology.

Papanicolaou stain

  • Abbreviated Pap stain.

Fontana-Masson stain

  • AKA Masson-Fontana stain,[64] Fontana-Masson stain for melanin, melanin stain.

Schmorl's stain

  • Stains melanin.
    • Similar to Fontana-Masson stain.

Notes:[65]

Martius scarlet blue stain

General

  • Stains connective tissue and fibrin.[66]
  • Abbreviated MSB.

Use:

Interpretation

  • Muscle and fibrin - red.
  • Nuclei = brown/black.
  • Collagen - blue.
  • Red blood cells - yellow.

Image:

Ref.:[67]

Picro-Mallory stain

General

  • Find fibrin.

Interpretation[68]

  • Fibrin = red.
  • Erythrocytes = yellow.
  • Connective tissue = blue.

Image:

Verhoeff-van Gieson stain

Verhoeff stain redirects here.
  • AKA Elastic van Gieson stain, abbreviated EVG.

General

  • Similar to Masson Trichrome & Verhoeff stain.[69]

Use:

  • Examine large blood vessels.[70]

Interpretation

  • Elastin = black.
  • Collagen = bright red.
  • Muscle = dull red.

Copper stain

General

Note:

  • Copper staining is a non-specific finding seen in many liver diseases; it is associated with impaired bile secretion.[71]

Interpretation

  • Copper = red granules.

Images:

Shikata stain

  • AKA Orcein stain for copper-protein.
  • AKA Shikata-Cu,[73]
  • AKA Shikata's orcein staining.[74]

General

Interpretation

Features:[75]

  • Purple/brown = elastin fibres.
  • Red = nuclei.
  • Light purple = background
  • ??? = Copper associated protein.

Gömöri Trichrome stain

  • Named after George Gömöri[76]

General

Interpretation

  • Dark green = muscle fibers.
  • Red = nuclei.
  • Bright red = mitochondria, red blood cells.

Images:

Miller stain

General

Interpretation

Staining:[77]

  • Black = elastin fibres, granules in mast cells.
  • Red = collagen.
  • Yellow = muscle, fibrin, erythrocytes.
  • Green/brown = nuclei.

See also

References

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