Stains

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

Where to start...

Principles

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

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

Main article: 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.

Basic panel:

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

Fungi

Main article: 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

General

• Abbreviated H&E.
• Standard bearer in most pathology departments.

Intepretation

• Blue (haematoxylin) = nucleus.
• Pink (eosin) = cytoplasm.

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

• 

  Perineural invasion - prostate - HPS stain (WC)

• 

  Meningioma - HPS stain (WC)

• 

  Endoneurial fibrosis - HPS stain (WC)

Periodic acid Schiff stain

• Abbreviated PAS.

Primary application

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

Utility

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

Interpretation

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

Ref.:

Image

• 

  Metanephric adenoma - PAS (WC)

Periodic acid Schiff with diastase

• Abbreviated: PAS-D and PASD.

General

• Diastase digests the glycogen.
• "PAS diastase resistant" 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:

Gomori methenamine-silver stain

• Abbreviated GMS.

Note:

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

Use

• Useful for fungi.
  • Pneumocystis jirovecii - cause of pneumocystis pneumonia (PCP).
  • Histoplasma - cause of histoplasmosis.
    • Histoplasma = black, round balls.

Image

• 

  GMS showing histoplasma (WC/Nephron)

Acid-fast bacilli stains

• Abbreviated: AFB.

There are several AFB stains:

• Ziehl-Neelson stain - used to look for Mycobacterium tuberculosis.
• Fite stain - used to look for Mycobacterium leprae.
• Auramine-rhodamine stain.

Ziehl-Neelsen stain

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

Image

• 

  ZN stain. (WC/CDC)

Fite stain

Interpretation:

• Red = AFB.
• Blue = background.

Auramine-rhodamine stain

• Fluorescent stain.

Image

• 

  AR stain. (WC/CDC)

Kinyoun stain

• Another AFB stain - useful for cryptosporidiosis and microsporidiosis.

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.
  • If the section is too thin... it doesn't work.

Interpretation

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

Ref.:

Image

• 

  Congo red staining in cerebral amyloid angiopathy. (WC)

Thioflavin T stain

Use

• Used to look for amyloid.

Interpretation

• Amyloid = green.

Image: Amyloid (inano.au.dk).

Gram stain

Use

• "It is useless for finding bacteria."
  • 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:

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
Cocci	Streptococcus pneumoniae, Staphylococcus aureus	Neisseria meningitidis, Moraxella catarrhalis

Interpretation

• Purple (or blue) = Gram positive organisms.
• Red = Gram negative organisms, nuclei.
• 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.
• Epithelial cell nuclei & stromal cell nuclei may stain red.
• Memory device: purple = positive.

Images

• 

  Gram positive cocci. (WC)

• 

  Gram positive rods - anthrax. (WC)

Luxol fast blue stain

• Abbreviated LFB.

Use

• Neuropathology, myelin stain.

Intepretation

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

Ref.:

Image

• 

  Globus pallidus and putamen - H&E-LFB. (WC)

Giemsa stain

Use

• Useful for finding mast cells.
• Useful for finding Donovan bodies and Leishmania.

Interpretation

• Tissue is light blue/green.

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:
  • Sarcomas have reticulin around each cell.
  • Carcinomas have reticulin around clusters of cells.

Interpretation

• Black = reticular fibers.
• Red = nuclei.

Notes:

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:

• Prussian blue stain - hemosiderin (vetmed.vt.edu).

Notes:

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

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.

Notes:

• PAS also stains lipofuscin and is more commonly available.

Interpretation

• Blue pigmented granules = lipofuscin.

Notes:

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

Oil red O stain

Use

• Stain adipose tissue.

Notes:

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

Warthin-Starry stain

Background:

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

Use

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

Interpretation:

• Spirochetes - black.
• Background - yellow.

Image

• 

  Helicobacter gastritis - Warthin-Starry stain. (WC)

Notes:

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

Dieterle stain

Considered a variant of the Steiner stain.

Use

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

Interpretation

• Spirochetes - black.
• Background - yellow.

Images

• 

  Dieterle stain - T. pallidum. (WC)

www:

• Treponema (med.sc.edu).
• Spirochetes - several images (med.sc.edu).

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.:

Image

• 

  Bielschowsky stain. (WC/Nephron)

Mucicarmine stain

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

Use

• Identify mucin.
• Malignant cells that produce mucin... carcinomas.

Interpretation

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

Images:

• Mucicarmine stained bowel (medschool.lsuhsc.edu).
• Mucicarmine stained pancreatic adenosquamous carcinoma (nature.com).

Alcian blue stain

General

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

Note:

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

Use

• Identify intestinal metaplasia in the stomach -- goblets = blue.

Interpretation

• Blue = acidic mucins.

Notes:

• Mucin stains:
  • Alcian blue stain, PASD stain, Mucicarmine stain.

Movat's stain

Use

• Myxomatous degeneration of cardiac valves.

Components

• Acid fuchsin, alcian blue, crocein scarlet, elastic hematoxylin, and saffron.

Interpretation of Movat stain

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

Reference:

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

Images

• 

  Cardiac amyloidosis - Movat stain. (WC/Nephron)

• 

  Cystic medial degeneration - Movat stain - low mag. (WC/Nephron)

• 

  Cystic medial degeneration - Movat stain - intermed. mag. (WC/Nephron)

Masson's trichrome stain

• Should not be confused with the Mallory trichrome stain.
• May be referred to as trichrome stain.

General

• Collagen vs. muscle.

Interpretation

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

Notes:

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

• Should not be confused with Masson trichrome stain.
• May be referred to as trichrome stain.

General

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

Site

• Kidney Bx (to assess for fibrosis).
  • Considered better than the Masson trichrome stain.
• 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

• 

  Cirrhosis. Mallory trichrome. (WC/Nephron)

Haematoxylin orcein phyloxin saffron stain

• Abbreviated HOPS.
• 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.
• AKA Methenamine PAS, abbreviated MPAS.

Use

• Visualize basement membrane in kidney biopsies.
  • Especially useful for the diagnosis of membranous nephropathy (MN).

Interpretation

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

Notes:

Images

• 

  MN demonstrated with a MPAS - very high mag. (WC/Nephron)

• 

  MN demonstrated with a MPAS - very high mag. (WC/Nephron)

Hale's colloidal iron stain

Use

• Chromophobe renal cell carcinoma vs. renal oncocytoma - chromophobe renal cell carcinoma +ve.

Notes:

Interpretation

• Blue (granular cytoplasmic) staining is positive.

Images:

• ChRCC Hale's colloidal iron (ultrapath.org).
• ChRCC Hale's colloidal iron (diagnosticpathology.org).

Notes:

• Often described as a "fastidious" (difficult/demanding) stain.
  • A few staff think this is a totally useless stain.

von Kossa stain

Use

• Look for calcium.

Interpretation

• Black = calcium.

Toluidine blue stain

Use

• May be useful in kidney biopsies.
• Stains mast cells, pneumocystis jirovecii.

Interpretation

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

Refs: looks a bit sketchy,

Image

• 

  PCP stained with toluidine blue. (WC)

www:

• Mast cells stained with toluidine blue (biomedcentral.com).

Romanowsky stain

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

Interpretation:

• 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.

Uses:

• Cytopathology.
• Helicobacter gastritis - organisms are dark blue against a light blue background.

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

• Often abbreviated Pap stain.
• Can be thought of as the H&E of cytopathology.
  • It is a modified H&E stain.
• Specimens are fixed in ethanol.
• Good for seeing nuclear detail.
• Out-of-focus cytoplasm is translucent; allows one to focus overlapped cells in different planes.

Use

• Cytopathology.

Interpretation

• Blue/purple = nucleus.
• Green/pink = cytoplasm.
• Orange = keratin.

Image=

• 

  Pap stain - urine cytology (WC)

Fontana-Masson stain

• AKA Masson-Fontana stain, Fontana-Masson stain for melanin, melanin stain.
• A type of silver stain.

Stains:

1. Melanin.
2. "Argentaffin granules" of the digestive tract.
3. Pigment deposition due to minocycline treatment.

Use

• Stain for melanin.
  • Used to differentiate brown pigments (lipofuscin, hemosiderin, melanin).
    • Many pathologists prefer IHC, i.e. Melan A over this stain.

Image:

• Fontana-Masson stain (vetmed.vt.edu).

Schmorl's stain

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

Notes:

Martius scarlet blue stain

General

• Stains connective tissue and fibrin.
• Abbreviated MSB.

Use:

• Look for fibrinoid necrosis in vasculitis.

Interpretation

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

Image:

• MSB (bris.ac.uk).

Ref.:

Picro-Mallory stain

General

• Find fibrin.

Interpretation

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

Image:

• Picro-Mallary (blogspot.com).

Verhoeff-van Gieson stain

• AKA Elastic van Gieson stain, abbreviated EVG.

General

• Similar to Masson Trichrome & Verhoeff stain.

Use:

• Examine large blood vessels.

Interpretation

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

Copper stain

General

• Used in liver biopsies.
• May be seen in Wilson's disease.

Note:

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

Interpretation

• Copper = red granules.

Images:

• Wilson's disease (naika.or.jp).

Shikata stain

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

General

• Used in medical liver biopsies - used to find copper.

Interpretation

Features:

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

See also

• Immunohistochemistry.
• Basics.

External links

• Procedure manuals - med.utah.edu.
• Special stains (introduction) - med.utah.edu.
• Stains - histology-world.com.