Difference between revisions of "Stains"

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m (→‎Dieterle stain: wikify syphilis)
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==Toluidine blue stain==
==Toluidine blue stain==
===Use===
===Use===
*May be useful in kidney biopsies.<ref name=pmid16799480>{{cite journal |author=Fischer EG, Moore MJ, Lager DJ |title=Fabry disease: a morphologic study of 11 cases |journal=Mod. Pathol. |volume=19 |issue=10 |pages=1295–301 |year=2006 |month=October |pmid=16799480 |doi=10.1038/modpathol.3800634 |url=http://www.nature.com/modpathol/journal/v19/n10/abs/3800634a.html}}</ref>
*May be useful in kidney biopsies.<ref name=pmid16799480>{{cite journal |author=Fischer EG, Moore MJ, Lager DJ |title=Fabry disease: a morphologic study of 11 cases |journal=Mod. Pathol. |volume=19 |issue=10 |pages=1295–301 |year=2006 |month=October |pmid=16799480 |doi=10.1038/modpathol.3800634 |url=http://www.nature.com/modpathol/journal/v19/n10/abs/3800634a.html}}</ref><ref name=pmid21659728>{{Cite journal  | last1 = Nicholas | first1 = SB. | last2 = Basgen | first2 = JM. | last3 = Sinha | first3 = S. | title = Using stereologic techniques for podocyte counting in the mouse: shifting the paradigm. | journal = Am J Nephrol | volume = 33 Suppl 1 | issue =  | pages = 1-7 | month =  | year = 2011 | doi = 10.1159/000327564 | PMID = 21659728 }}</ref>
*Stains [[mast cell]]s, [[pneumocystis jirovecii]].
*Stains [[mast cell]]s, [[pneumocystis jirovecii]].



Revision as of 02:59, 30 July 2011

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 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

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).

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 use

  • Collagen vs. muscle.

Site

  • Kidney Bx (to assess for fibrosis).
  • Liver Bx (to assess for cirrhosis).

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.

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

Interpretation

  • Blue is positive.[6]

Notes:

  • A few staff think this is a totally useless stain.[40]

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.

Refs: looks a bit sketchy[43], [44]

Image:

Romanowsky stain

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


Interpretation:[45]

  • 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.[46]
  • 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).[47]
      • Many pathologists prefer IHC, i.e. Melan A over this stain.

Image:

Schmorl's stain

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

Notes:[48]

Martius scarlet blue stain

General

  • Stains connective tissue and fibrin.[49]
  • Abbreviated MSB.

Use:

Interpretation

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

Image:

Ref.:[50]

Picro-Mallory stain

General

  • Find fibrin.

Interpretation[51]

  • 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.[52]

Use:

  • Examine large blood vessels.[53]

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.[54]

Interpretation

  • Copper = red granules.

Images:

Shikata stain

  • AKA Orcein stain for copper-protein, AKA Shikata-Cu,[56] AKA Shikata's orcein staining.[57]

General

Interpretation

Features:[58]

  • 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. http://library.med.utah.edu/WebPath/HISTHTML/MANUALS/MASSONS.PDF
  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. ALS several occasions - 2009.
  41. 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.
  42. 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.
  43. URL: http://www.molecularstation.com/protocol-links/articles/Toluidine-Blue-Stain-32.html. Accessed on: 17 March 2011.
  44. URL: http://www.dermnetnz.org/doctors/dermatopathology/stains.html. Accessed on: 17 March 2011.
  45. 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/.
  46. URL: http://www.ihcworld.com/_protocols/special_stains/diff_quick_ellis.htm. Accessed on: 4 January 2010.
  47. URL: http://education.vetmed.vt.edu/curriculum/VM8054/labs/Lab2/Examples/exfontana.htm. Accessed on: 5 May 2010.
  48. URL: http://library.med.utah.edu/WebPath/HISTHTML/STAINS/STAINS.html. Accessed on: 5 May 2010.
  49. URL: http://www.bris.ac.uk/vetpath/cpl/msb.html. Accessed on: 26 November 2010.
  50. URL: http://www.bris.ac.uk/vetpath/cpl/msb.html. Accessed on: 26 November 2010.
  51. "Picro-Mallory for Fibrin – Long Version". http://stainsfile.info/StainsFile/stain/fibrin/picro-mallory-1.htm. Retrieved 17 January 2011.
  52. URL: http://education.vetmed.vt.edu/Curriculum/VM8054/Labs/Lab2/Examples/exvrmass.htm. Accessed on: 3 January 2011.
  53. URL: http://education.vetmed.vt.edu/Curriculum/VM8054/Labs/Lab2/Examples/exvvg.htm. Accessed on: 3 January 2011.
  54. 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.
  55. URL: http://www.naika.or.jp/im2/42/10/14c.aspx. Accessed on: 24 January 2011.
  56. http://www.mayomedicallaboratories.com/test-catalog/Overview/9836. Accessed on: 24 January 2011.
  57. URL: http://informahealthcare.com/doi/abs/10.3109/00313027709085239?journalCode=pat. Accessed on: 24 January 2011.
  58. URL: http://www.nottingham.ac.uk/pathology/protocols/shikata.html. Accessed on: 24 January 2011.

External links