Stains

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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]

  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.[2]

Basic panel:

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

Fungi

Main article: Microorganisms

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.
  • 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.

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.

Image: Metanephric adenoma - PAS (WC).

Ref.:[4]

Periodic acid Schiff with diastase

  • Abbreviated: PAS-D and PASD.

General

  • Diastase digests the glycogen.

Use

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

Notes: [5]

Gomori methenamine-silver stain

  • Abbreviated GMS.

Note:

  • GMS is "Grocott's methenamine Silver" according to WMSP.[6]

Use

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

Image: GMS showing histoplasma (WC).

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 laprae.[7]
  • Auramine-rhodamine stain.

Ziehl-Neelsen stain

  • Most popular acid-fast bacilli stain.
  • Stains other mycobacteria -- not specific for tuberculosis.
    • Stains nocardia.[8]

Image: ZN stain (WC/CDC).

Fite stain

Interpretation:

  • Red = AFB.
  • Blue = background.

Auramine-rhodamine stain

  • Fluorescent stain.

Image: AR stain (WC/CDC).

Congo red stain

Use

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

Interpretation

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

Ref.:[9]

Image: Congo red stain - cerebral amyloid angiopathy (WC).

Thioflavin T stain

Use

Interpretation

  • Amyloid = green.

Image: Amyloid (inano.au.dk).

Gram stain

Use:

  • "It is useless for finding bacteria."[10]
    • I suppose this is as you find 'em on H&E... or microbiology finds 'em.

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.[11]
  • Epithelial cell nuclei & stromal cell nuclei may stain red.
  • Memory device: purple = positive.

Ref.:[12]

Luxol fast blue stain

  • Abbreviated LFB.

Use

  • Neuropathology, myelin stain.

Intepretation

  • Blue = myelinated fibers (contain lipoproteins), lipofuscin.[13]
    • Lack of blue (where it ought to be) = demyelination.
  • Purple = nerve cell (e.g. neuron).
  • Neutrophils = pink.

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

Ref.:[14]

Giemsa stain

Use

  • Useful for finding mast cells.
  • Useful for finding donovan bodies and leishmania.[15]

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:[16]
    • Sarcomas have reticulin around each cell.
    • Carcinomas have reticulin around clusters of cells.

Interpretation

  • Black = reticular fibers.
  • Red = nuclei.

Notes:[17]

Cresyl violet stain

  • Everything is shades of blue.
  • Used at some places (e.g. SMH) to look for Helicobacter organisms.
    • Helicobacter stains blue.

Prussian blue stain

  • AKA Perl's iron stain.
  • Useful for iron and hemosiderin; useful for differentiating brown pigments (melanin, lipofuscin, tattoo pigment, hemosiderin).

Image:

Notes:

  • Described well by vetmed.vt.edu.[18]
  • 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.[19]
  • 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.[20]

Use

  • Find spirochetes, e.g. syphilis (Treponema pallidum),[21] cat-scratch disease (Bartonella henselae).
  • Find Helicobacter spp., e.g. Helicobacter pylori -- Mount Sinai Hospital.[22]

Interpretation:[23]

  • Spirochetes - black.
  • Background - yellow.

Image:

Notes:

  • Considered a "dirty" stain - picks-up junk in the background.[24]

Dieterle stain

Considered a variant of the Steiner stain.[25]

Use

  • Find spirochetes, e.g. syphilis (Treponema pallidum),[26] donovan bodies (leishmaniasis),[27] Helicobacter pylori and Bartonella henselae (Cat-scratch disease).[28]

Interpretation

  • Spirochetes - black.
  • Background - yellow.

Images: Dieterle stain - T. pallidum (WC), 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.

Image: Bielschowsky stain (WC).

Ref.: [29]

Mucicarmine stain

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

Use

  • Identify mucin.
  • Malignant cells that produce mucin... carcinomas.[30]

Interpretation

  • Carmine with metanil yellow and Weigert's Hematoxylin:[31]
    • Blue/black = nucleus.
    • Yellow = background.
    • Red = mucin.[32]

Images:

Alcian blue

General

  • Stains acidic mucin (pH=2.5); Alcian blue = Acidic.
    • A variant uses pH=1.0.[6]

Use

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

Interpretation

  • Blue = acidic mucins.

Notes:[33]

Movat's stain

Use

Components

  • Acid fuchsin, alcian blue, crocein scarlet, elastic hematoxylin, and saffron.[34][35]

Interpretation of Movat stain

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

Reference: [35]

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

Masson's trichrome stain

General

  • Collagen vs. muscle.

Interpretation

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

Notes: [36]

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:

HOPS stain

  • The name is an acronym for... haematoxylin, orcein, phyloxin and saffron.[37]
  • It should not be confused with the HPS stain.

Interpretation

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

Jones stain

Use

  • Visualize basement membrane in kidney biopsies.

Interpretation

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

Notes:[38]

Hale's colloidal iron stain

Use

Notes:

Interpretation

  • Blue (granular cytoplasmic) staining is positive.[6]

Images:

Notes:

  • Often described as a "fastidious" (difficult/demanding) stain.[41]
    • A few staff think this is a totally useless stain.[42]

von Kossa stain

Use

  • Look for calcium.

Interpretation

  • Black = calcium.[6]

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[45], [46]

Image:

Romanowsky stain

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


Interpretation:[47]

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

Field's 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

  • Proprietary variant of Romanowsky stain.[48]
  • Use: cytopathology.

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:

Fontana-Masson stain

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

Stains:

  1. Melanin.
  2. "argentaffin granules" of the digestive tract.

Use

  • Stain for melanin.
    • Used to differentiate brown pigments (lipofuscin, hemosiderin, melanin).[49]
      • Many pathologists prefer IHC, i.e. Melan A over this stain.

Image:

Schmorl's stain

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

Notes:[50]

Martius scarlet blue stain

General

  • Stains connective tissue and fibrin.[51]
  • Abbreviated MSB.

Use:

Interpretation

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

Image:

Ref.:[52]

Picro-Mallory stain

General

  • Find fibrin.

Interpretation[53]

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

Image:

Verhoeff-van Gieson stain

General

  • AKA Elastic van Gieson stain.
    • Abbreviated EVG.
  • Similar to Masson Trichrome & Verhoeff stain.[54]

Use:

  • Examine large blood vessels.[55]

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.[56]

Interpretation

  • Copper = red granules.

Images:

Shikata stain

  • AKA Orcein stain for copper-protein, AKA Shikata-Cu,[58] AKA Shikata's orcein staining.[59]

General

Interpretation

Features:[60]

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

See also

References

  1. ↑ LAE. 13 July 2010.
  2. ↑ Woods GL, Walker DH (July 1996). "Detection of infection or infectious agents by use of cytologic and histologic stains". Clin. Microbiol. Rev. 9 (3): 382-404. PMC 172900. PMID 8809467. http://cmr.asm.org/cgi/pmidlookup?view=long&pmid=8809467.
  3. ↑ Kovi J, Leifer C (July 1970). "Lipofuscin pigment accumulation in spontaneous mammary carcinoma of A/Jax mouse". J Natl Med Assoc 62 (4): 287–90. PMC 2611776. PMID 5463681. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2611776/pdf/jnma00512-0077.pdf.
  4. ↑ http://library.med.utah.edu/WebPath/HISTHTML/MANUALS/PAS.PDF
  5. ↑ http://library.med.utah.edu/WebPath/HISTHTML/MANUALS/PASD.PDF
  6. ↑ 6.0 6.1 6.2 6.3 Humphrey, Peter A; Dehner, Louis P; Pfeifer, John D (2008). The Washington Manual of Surgical Pathology (1st ed.). Lippincott Williams & Wilkins. pp. 682. ISBN 978-0781765275.
  7. ↑ URL: http://library.med.utah.edu/WebPath/HISTHTML/MANUALS/FITES.PDF. Accessed on: 19 May 2011.
  8. ↑ URL: http://library.med.utah.edu/WebPath/LUNGHTML/LUNG024.html. Accessed on: 19 May 2011.
  9. ↑ URL: http://library.med.utah.edu/WebPath/HISTHTML/MANUALS/CONGORED.PDF. Accessed on: 4 December 2010.
  10. ↑ SMH handout.
  11. ↑ URL: http://www.lpi.usra.edu/publications/slidesets/marslife/slide_27.html.
  12. ↑ http://library.med.utah.edu/WebPath/HISTHTML/MANUALS/GRAM.PDF
  13. ↑ MUN. 26 November 2010.
  14. ↑ http://library.med.utah.edu/WebPath/HISTHTML/MANUALS/LFB.PDF
  15. ↑ URL: http://library.med.utah.edu/WebPath/HISTHTML/STAINS/STAINS.html. Accessed on: April 6, 2009.
  16. ↑ MACKENZIE DH (March 1958). "Reticulin patterns in the diagnosis of carcinomas and sarcomas". Br. J. Cancer 12 (1): 14–9. PMC 2074006. PMID 13536209. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2074006/.
  17. ↑ http://library.med.utah.edu/WebPath/HISTHTML/MANUALS/RETIC.PDF
  18. ↑ Prussian blue stain. URL:[http://education.vetmed.vt.edu/curriculum/VM8054/labs/Lab2/Examples/exprussb.htm. Accessed on: 5 May 2010.
  19. ↑ Kluver-Barrera stain. URL:http://education.vetmed.vt.edu/curriculum/VM8054/labs/Lab2/Examples/exkluvbarr.htm. Accessed on: 5 May 2010.
  20. ↑ URL: http://www.merriam-webster.com/medical/warthin. Accessed on: 17 August 2010.
  21. ↑ URL: http://library.med.utah.edu/WebPath/HISTHTML/STAINS/STAINS.html. Accessed on: April 6, 2009.
  22. ↑ http://www.dako.co.uk/index/prod_search/prod_products.htm?productareaid=41&baseprodidver=A224462007
  23. ↑ http://library.med.utah.edu/WebPath/HISTHTML/STAINS/STAIN029.html
  24. ↑ DB. 4 August 2010.
  25. ↑ URL: http://www.mayomedicallaboratories.com/test-catalog/Overview/80327. Accessed on: 8 August 2010.
  26. ↑ Humphrey, Peter A; Dehner, Louis P; Pfeifer, John D (2008). The Washington Manual of Surgical Pathology (1st ed.). Lippincott Williams & Wilkins. pp. 455. ISBN 978-0781765275.
  27. ↑ URL: http://www.mondofacto.com/facts/dictionary?Dieterle%27s+stain. Accessed on: 4 August 2010.
  28. ↑ URL: http://www.mayomedicallaboratories.com/test-catalog/Overview/80327. Accessed on: 8 August 2010.
  29. ↑ http://library.med.utah.edu/WebPath/HISTHTML/MANUALS/BIELSCH.PDF
  30. ↑ Lefkowitch, Jay H. (2006). Anatomic Pathology Board Review (1st ed.). Saunders. pp. 681 (Q25). ISBN 978-1416025887.
  31. ↑ Humphrey, Peter A; Dehner, Louis P; Pfeifer, John D (2008). The Washington Manual of Surgical Pathology (1st ed.). Lippincott Williams & Wilkins. pp. 678. ISBN 978-0781765275.
  32. ↑ http://www.medschool.lsuhsc.edu/pathology/pathist/SURGPATH/special%20stains/assets/mucicarmine3.jpg
  33. ↑ http://library.med.utah.edu/WebPath/HISTHTML/MANUALS/ALCIAN.PDF
  34. ↑ [1]
  35. ↑ 35.0 35.1 Modified Movat's Pentachrome Stain. University Penn Medicine. URL: http://www.med.upenn.edu/mcrc/histology_core/movat.shtml. Accessed on: January 29, 2009.
  36. ↑ ULR: http://library.med.utah.edu/WebPath/HISTHTML/MANUALS/MASSONS.PDF. Accessed on: 2 November 2011.
  37. ↑ Perry JR, Bilbao JM, Gray T (1992). "Fatal basilar vasculopathy complicating bacterial meningitis". Stroke 23 (8): 1175–8. PMID 1636194. Free Full Text.
  38. ↑ URL: http://library.med.utah.edu/WebPath/HISTHTML/MANUALS/JONES.PDF. Accessed on: 19 May 2011.
  39. ↑ Tickoo SK, Amin MB, Zarbo RJ (April 1998). "Colloidal iron staining in renal epithelial neoplasms, including chromophobe renal cell carcinoma: emphasis on technique and patterns of staining". Am. J. Surg. Pathol. 22 (4): 419–24. PMID 9537468. http://meta.wkhealth.com/pt/pt-core/template-journal/lwwgateway/media/landingpage.htm?issn=0147-5185&volume=22&issue=4&spage=419.
  40. ↑ URL: http://www.ultrapath.org/oldsite/cases99/sep99/cotm9-2.html. Accessed on: 9 October 2011.
  41. ↑ URL: http://www.merriam-webster.com/dictionary/fastidious?show=0&t=1319550566. Accessed on: 25 October 2011.
  42. ↑ ALS. On several occasions in 2009.
  43. ↑ Fischer EG, Moore MJ, Lager DJ (October 2006). "Fabry disease: a morphologic study of 11 cases". Mod. Pathol. 19 (10): 1295–301. doi:10.1038/modpathol.3800634. PMID 16799480. http://www.nature.com/modpathol/journal/v19/n10/abs/3800634a.html.
  44. ↑ Nicholas, SB.; Basgen, JM.; Sinha, S. (2011). "Using stereologic techniques for podocyte counting in the mouse: shifting the paradigm.". Am J Nephrol 33 Suppl 1: 1-7. doi:10.1159/000327564. PMID 21659728.
  45. ↑ URL: http://www.molecularstation.com/protocol-links/articles/Toluidine-Blue-Stain-32.html. Accessed on: 17 March 2011.
  46. ↑ URL: http://www.dermnetnz.org/doctors/dermatopathology/stains.html. Accessed on: 17 March 2011.
  47. ↑ Horobin RW, Walter KJ (1987). "Understanding Romanowsky staining. I: The Romanowsky-Giemsa effect in blood smears". Histochemistry 86 (3): 331–6. PMID 2437082. http://www.springerlink.com/content/r81x25451m841866/.
  48. ↑ URL: http://www.ihcworld.com/_protocols/special_stains/diff_quick_ellis.htm. Accessed on: 4 January 2010.
  49. ↑ URL: http://education.vetmed.vt.edu/curriculum/VM8054/labs/Lab2/Examples/exfontana.htm. Accessed on: 5 May 2010.
  50. ↑ URL: http://library.med.utah.edu/WebPath/HISTHTML/STAINS/STAINS.html. Accessed on: 5 May 2010.
  51. ↑ URL: http://www.bris.ac.uk/vetpath/cpl/msb.html. Accessed on: 26 November 2010.
  52. ↑ URL: http://www.bris.ac.uk/vetpath/cpl/msb.html. Accessed on: 26 November 2010.
  53. ↑ "Picro-Mallory for Fibrin – Long Version". http://stainsfile.info/StainsFile/stain/fibrin/picro-mallory-1.htm. Retrieved 17 January 2011.
  54. ↑ URL: http://education.vetmed.vt.edu/Curriculum/VM8054/Labs/Lab2/Examples/exvrmass.htm. Accessed on: 3 January 2011.
  55. ↑ URL: http://education.vetmed.vt.edu/Curriculum/VM8054/Labs/Lab2/Examples/exvvg.htm. Accessed on: 3 January 2011.
  56. ↑ Miyamura H, Nakanuma Y, Kono N (December 1988). "Survey of copper granules in liver biopsy specimens from various liver abnormalities other than Wilson's disease and biliary diseases". Gastroenterol. Jpn. 23 (6): 633–8. PMID 2464523.
  57. ↑ URL: http://www.naika.or.jp/im2/42/10/14c.aspx. Accessed on: 24 January 2011.
  58. ↑ http://www.mayomedicallaboratories.com/test-catalog/Overview/9836. Accessed on: 24 January 2011.
  59. ↑ URL: http://informahealthcare.com/doi/abs/10.3109/00313027709085239?journalCode=pat. Accessed on: 24 January 2011.
  60. ↑ URL: http://www.nottingham.ac.uk/pathology/protocols/shikata.html. Accessed on: 24 January 2011.

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