Difference between revisions of "Immunohistochemical staining"

From Libre Pathology
Jump to navigation Jump to search
(wikify INI1 p63 STAT6)
(→‎Interpretation: base error rate)
Line 40: Line 40:
#*A combination of the above.
#*A combination of the above.


Generally, interpretations can be subjective, and this is especially true when the staining is weak and focal. In other words, "... your weak [positive] stain might be somebody else’s negative."<ref>URL: [http://bitesizebio.com/articles/immunohistochemistry-getting-the-stain-you-want/ http://bitesizebio.com/articles/immunohistochemistry-getting-the-stain-you-want/]. Accessed on: 1 September 2012.</ref>
Generally, interpretations can be subjective, and this is especially true when the staining is weak and focal. In other words, "... your weak [positive] stain might be somebody else’s negative."<ref>URL: [http://bitesizebio.com/articles/immunohistochemistry-getting-the-stain-you-want/ http://bitesizebio.com/articles/immunohistochemistry-getting-the-stain-you-want/]. Accessed on: 1 September 2012.</ref> This is often reflected in publications repoting contradictory results regarding the rates of positivity for stains in different tumours, even if the medthods uses are identical.


The cynical might say it is unwritten rule that: "... if the stain is weak and focal it can be anything you want to make it -- positive or negative -- so it fits perfectly with your diagnosis!"
The cynical might say it is unwritten rule that: "... if the stain is weak and focal it can be anything you want to make it -- positive or negative -- so it fits perfectly with your diagnosis!"


In cases where the morphology is unclear, it is judicious to have two or more immunostains that support the diagnosis, and negative stains for important entities in the differential diagnosis.
In cases where the morphology is unclear, it is judicious to have two or more immunostains that support the diagnosis, and negative stains for important entities in the differential diagnosis.
One third pathologists substantially overestimate and one third underestimate the diagnostic significance of unexpected immunohistochemical staining results.<ref>{{Cite journal  | last1 = Galloway | first1 = M. | title = Base-rate error in the interpretation of immunohistochemistry. | journal = Patholog Res Int | volume = 2011 | issue =  | pages = 636495 | month =  | year = 2011 | doi = 10.4061/2011/636495 | PMID = 21660231 }}</ref>


==General (malignant) differential diagnosis==
==General (malignant) differential diagnosis==

Revision as of 14:38, 24 September 2015

CK20 staining of normal small intestinal mucosa.

Immunohistochemical staining, also immunostaining, is a powerful tool. It is abbreviated IHC.

Utility

Use of immunohistochemistry:[1]

  1. Unknown primary tumours.
  2. Poorly differentiated tumours.
  3. Prognostic markers, e.g. ERBB2 (HER2).
  4. Proving clonality - in the context of hematologic malignancies.

Theory

  • Antigen retrieval.
  • Antibody binds to the antigen.
  • Amplification - needed as the signal is usually too weak.

Signaling systems

  • Avidin-biotin complex.
    • Older.[2]
    • May suffer from endogenous avidin-biotin activity.[3]
  • Polymer based methods.
    • Newer.

Quality control

This is an evolving area in pathology that has been ignored for a surprisingly long time.

It is touched upon the in the quality article in the immunohistochemistry section.

Interpretation

To determine whether a stain is (1) done correctly, and (2) positive, one needs to know:

  1. What tissues it stains:
    • Tumour.
    • Normal tissue.
  2. How it stains the various tissues:
    • Patchy.
    • Diffuse.
  3. Where it stains the various tissues:
    • Nucleus.
    • Cytoplasm.
    • Membrane.
    • A combination of the above.

Generally, interpretations can be subjective, and this is especially true when the staining is weak and focal. In other words, "... your weak [positive] stain might be somebody else’s negative."[4] This is often reflected in publications repoting contradictory results regarding the rates of positivity for stains in different tumours, even if the medthods uses are identical.

The cynical might say it is unwritten rule that: "... if the stain is weak and focal it can be anything you want to make it -- positive or negative -- so it fits perfectly with your diagnosis!"

In cases where the morphology is unclear, it is judicious to have two or more immunostains that support the diagnosis, and negative stains for important entities in the differential diagnosis.

One third pathologists substantially overestimate and one third underestimate the diagnostic significance of unexpected immunohistochemical staining results.[5]

General (malignant) differential diagnosis

 
 
 
 
 
 
 
 
 
 
Malignancy
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Epithelial
(Carcinoma)
 
Mesenchymal
(Sarcoma)
 
Germ cell
tumour
 
Neuroendocrine
carcinoma
 
Hematologic
 
Malignant
melanoma
  • Carcinoma.
    • AE1/AE3 - pankeratin.
    • Others: EMA, HMWK, LMWK.
  • Sarcoma.
    • Vimentin.
      • Many pathologists think this stain is totally useless.
  • Germ cell tumours.
  • Neuroendocrine carcinoma.
    • Chromogranin A.
    • Synaptophysin.
    • CD56.
    • CD57.[7]
  • Melanoma.
    • S-100, HMB-45, Melan A (MART-1).
      • Additional: melanoma cocktail (HMB-45, MART-1)[8], microphthalmia,[9] tyrosinase.[10]
  • Hematologic.
    • Lymphoma/leukemia.
      • CD45 (common leukocyte antigen).
      • CD30.
    • Plasma cell:
      • Kappa.
      • Lambda.
      • CD138.

Keratins

Classification:[11]

  • Low molecular weight keratins (LMWK): 7, 8/18, 19, 20.
  • High molecular weight keratins (HWMK): 4, 10, 13, 14, 17.

Uses:

Organ specific

Thyroid and lung

Image: Adenocarcinoma with nuclear TTF-1 positivity (WC).

Breast markers

  • GCDFP-15 (AKA BRST-2) -- specific, but NOT sensitive.
  • ER (estrogen receptor) - in normal breast.
  • PR (progesterone receptor) - in normal breast.
  • HER2/neu - pathological, assoc. with worse prognosis.

Prostate gland

  • PSA - prostatic-specific antigen.
  • PSAP - prostatic-specific acid phosphatase.
  • p63 - stains nuclei of basal cell in normal prostate.
  • 34betaE12 - stains basal cells in normal prostate.
  • AMACR (racemase, P504S[18]) - present in adenocarcinoma (NOT in normal prostate).
  • AR - usually present in prostate confined cancers.[19]
  • CAP cocktail - AKA CAP, AKA PIN-4, AKA PIN.

Colorectal carcinoma markers

  • CK20.
  • CDX2.
    • Uncommon in primary lung, breast, pancreas, kidney, gallbladder, liver, urinary bladder, thyroid gland.[20]
  • CEA.

Small bowel

  • CDX2.
  • Villin.

Kidney

Renal cell carcinoma:

  • RCC, EMA, CD10.
  • CK7 -ve in clear cell RCC.
  • AMACR +ve in papillary RCC.
  • D2-40 +ve in ChRCC.

Xanthogranulomatous pyelonephritis:

  • CD68 (for macrophages).

Ovary

  • CA125, CK7+, CK20-.
  • WT1 -- 90% in serous +ve.

Serous markers

  • WT-1, CA-125, D2-40.

Liver

HCC vs. cholangiocarcinoma:

  • TTF-1: ~90-100% +ve (cytoplasmic) in HCC vs. ~10% in cholangiocarcinoma.[23]

Mesothelium

Panel:[24]

  • Mesothelial markers:
    • Calretinin.
    • WT-1.
    • D2-40.
    • CK5/6.
  • Carcinoma markers:
    • CEA (monoclonal and polyclonal).
    • TTF-1.
    • Ber-EP4.
    • MOC-31.

Note:

  • One should use two mesothelial markers and two carcinoma markers.[24]

Pancreas

Neuropathology

General:

  • S-100.

Glial:

  • GFAP.

Neuronal:

  • Synaptophysin.
  • Chromogranin.

Glial/Neuronal:

Specific entities:

Tumour (low-grade gliomas):

Miscellaneous

Macrophages

  • CD68.

Special:

Special, less common:

One organ versus another

Cervix versus uterus

  • Cervix (typically): CEA +ve,[33] p16 +ve.
    • ... and ER -ve, PR -ve, vimentin -ve.
  • Uterus (typically): vimentin +ve, ER +ve, PR +ve.[34]
    • ... and CEA -ve, p16 -ve.

Liver versus bile duct

Intrahepatic cholangiocarcinoma (ICC) vs. hepatocellular carcinoma (HCC):[35]

  • ICC: CK19 (92.5%), MUC-1 (73.8%) +ve.
  • HCC: HepPar-1 (85.6%), CD34 (87.8%) +ve.

Prostate versus bladder

Prostate adenocarcinoma vs. urothelial carcinoma:

  • Prostate adenocarcinoma: PSA +ve, PSAP +ve, AR +ve, CK7 -ve, CK20 -ve.
  • Urothelial carcinoma: CK7 +ve, CK20 +ve, PSA -ve, PSAP -ve, AR -ve.

Breast versus ovary

Breast carcinoma versus ovarian carcinoma:

Lymphomas

This is covered more extensively in the lymphoma article.

Lymphocytes

  • CD45 (AKA common leukocyte antigen).

B-cells

  • CD20.
  • CD19 (flow only).
  • PAX5.
  • CD79a.

T-cells

  • CD3 - general T-cell marker (marks both CD4 +ve and CD8 +ve cells).
  • CD4.
  • CD8.
  • CD7.
  • CD43.

Specific entities

Follicular lymphoma

  • CD10 +ve, BCL6 +ve.

CLL

Mantle cell lymphoma

Hodgkin's lymphoma

This is covered more extensively in the Hodgkin lymphoma article.

Classic types:

  • CD30 Reed-Sternberg cells (RSCs) +ve ~98%.[39]
  • CD15 Reed-Sternberg cells +ve ~80%, stains neutrophils.

Germ cell tumours

Seminoma

  • D2-40 +ve.[6]
  • OCT 3/4 +ve.

Embryonal carcinoma

  • CD30 +ve - cytoplasm, cell membrane, Golgi.
    • Rarely positive in seminoma.
  • CK7 +ve.[40]
  • AE1/AE3 +ve.

Yolk sac tumour (endodermal sinus tumour)

  • AFP (alpha fetoprotein).

Choriocarcinoma

  • beta-hCG.

Bare bones mnemonic for GCTs

The germ cell tumour (GCT) IHC mnemonic ABCD:

  • AFP = yolk sac tumour.
  • Beta-hCG = choriocarcinoma.
  • CD30 = embryonal carcinoma.
  • D2-40 = seminoma.

Spindle cell lesions

Abbreviated spindle cell panel (memory device SCADS):

  • S100.
  • CD34.
  • AE1/AE3.
  • Desmin.
  • SMA.

A MFH panel:

Muscle markers

  • Desmin - all three types.
  • H-caldesmon - smooth muscle - most specific.
  • Smooth muscle actin (SMA) - smooth muscle.
  • MyoD1 - skeletal muscle.
  • Smooth muscle myosin (abbreviated SMMS).

Proliferation markers

  • MIB1 - an antibody against the protein Ki-67 (a protein expressed in proliferating cells).

Notes:

  • Ki-67 is found in lymphocytes - these are a positive control; lymphocytes should not be confused for (positive) tumour cells.[43]
  • MIB1 should not be confused with mindbomb homolog 1 (MIB-1), a gene that regulates apoptosis.[44]

Carcinomas

CK7 and CK20

CK7+ CK20-

Mnemonic: OBE + lung x2 + H&N x2

CK7- CK20+

CK7+ CK20+

Mnemonic: POOTE.

Note:

CK7- CK20-

Mnemonic: NASH TURP.

Vimentin and cytokeratin positivity

A few tumours are positive for both vimentin and cytokeratins.

Common tumours:

Rare tumours:

Common tumours that uncommonly have the pattern:

Sarcomas cytokeratins

Most sarcomas are cytokeratin negative.

Exceptions - classic:

Others:

See also

References

  1. Mitchell, Richard; Kumar, Vinay; Fausto, Nelson; Abbas, Abul K.; Aster, Jon (2011). Pocket Companion to Robbins & Cotran Pathologic Basis of Disease (8th ed.). Elsevier Saunders. pp. 175. ISBN 978-1416054542.
  2. Vosse, BA.; Seelentag, W.; Bachmann, A.; Bosman, FT.; Yan, P. (Mar 2007). "Background staining of visualization systems in immunohistochemistry: comparison of the Avidin-Biotin Complex system and the EnVision+ system.". Appl Immunohistochem Mol Morphol 15 (1): 103-7. PMID 17536316.
  3. Vosse, BA.; Seelentag, W.; Bachmann, A.; Bosman, FT.; Yan, P. (Mar 2007). "Background staining of visualization systems in immunohistochemistry: comparison of the Avidin-Biotin Complex system and the EnVision+ system.". Appl Immunohistochem Mol Morphol 15 (1): 103-7. PMID 17536316.
  4. URL: http://bitesizebio.com/articles/immunohistochemistry-getting-the-stain-you-want/. Accessed on: 1 September 2012.
  5. Galloway, M. (2011). "Base-rate error in the interpretation of immunohistochemistry.". Patholog Res Int 2011: 636495. doi:10.4061/2011/636495. PMID 21660231.
  6. 6.0 6.1 Iczkowski KA, Butler SL, Shanks JH, et al (February 2008). "Trials of new germ cell immunohistochemical stains in 93 extragonadal and metastatic germ cell tumors". Hum. Pathol. 39 (2): 275-81. doi:10.1016/j.humpath.2007.07.002. PMID 18045648.
  7. Kurokawa, M.; Nabeshima, K.; Akiyama, Y.; Maeda, S.; Nishida, T.; Nakayama, F.; Amano, M.; Ogata, K. et al. (May 2003). "CD56: a useful marker for diagnosing Merkel cell carcinoma.". J Dermatol Sci 31 (3): 219-24. PMID 12727026.
  8. Jani P, Chetty R, Ghazarian DM (April 2008). "An unusual composite pilomatrix carcinoma with intralesional melanocytes: differential diagnosis, immunohistochemical evaluation, and review of the literature". Am J Dermatopathol 30 (2): 174–7. doi:10.1097/DAD.0b013e318165b8fe. PMID 18360125.
  9. URL: http://www.ncbi.nlm.nih.gov/omim/156845. Accessed on: 18 August 2010.
  10. Roma, AA.; Magi-Galluzzi, C.; Zhou, M. (Jan 2007). "Differential expression of melanocytic markers in myoid, lipomatous, and vascular components of renal angiomyolipomas.". Arch Pathol Lab Med 131 (1): 122-5. doi:10.1043/1543-2165(2007)131[122:DEOMMI]2.0.CO;2. PMID 17227112.
  11. http://www.nordiqc.org/Epitopes/Cytokeratins/cytokeratins.htm
  12. Murata T, Nakashima Y, Takeuchi M, Sueishi K, Inomata H (September 1993). "The diagnostic use of low molecular weight keratin expression in sebaceous carcinoma". Pathol. Res. Pract. 189 (8): 888–93. PMID 7508102.
  13. URL: http://www.aruplab.com/guides/ug/tests/2003978.jsp. Accessed on: 17 March 2011.
  14. Jagirdar J (March 2008). "Application of immunohistochemistry to the diagnosis of primary and metastatic carcinoma to the lung". Arch. Pathol. Lab. Med. 132 (3): 384–96. PMID 18318581. http://journals.allenpress.com/jrnlserv/?request=get-abstract&issn=0003-9985&volume=132&page=384.
  15. Dralle, H.; Böcker, W. (1982). "[Thyroglobulin immunohistochemistry: new aspects of pathophysiology and differential diagnosis of benign and malignant goitre (author's transl)].". Langenbecks Arch Chir 356 (3): 205-12. PMID 7070163.
  16. de Micco, C.; Chapel, F.; Dor, AM.; Garcia, S.; Ruf, J.; Carayon, P.; Henry, JF.; Lebreuil, G. (Mar 1993). "Thyroglobulin in medullary thyroid carcinoma: immunohistochemical study with polyclonal and monoclonal antibodies.". Hum Pathol 24 (3): 256-62. PMID 8454270.
  17. Azumi, N.; Traweek, ST.; Battifora, H. (Aug 1991). "Prostatic acid phosphatase in carcinoid tumors. Immunohistochemical and immunoblot studies.". Am J Surg Pathol 15 (8): 785-90. PMID 1712549.
  18. http://www.antibodies-online.com/antibody/125649/P504S+alphaMethylacylCoA+Racemace+AMACR+Human/
  19. Fleischmann, A.; Rocha, C.; Schobinger, S.; Seiler, R.; Wiese, B.; Thalmann, GN. (Apr 2011). "Androgen receptors are differentially expressed in Gleason patterns of prostate cancer and down-regulated in matched lymph node metastases.". Prostate 71 (5): 453-60. doi:10.1002/pros.21259. PMID 20878946.
  20. Kaimaktchiev, V.; Terracciano, L.; Tornillo, L.; Spichtin, H.; Stoios, D.; Bundi, M.; Korcheva, V.; Mirlacher, M. et al. (Nov 2004). "The homeobox intestinal differentiation factor CDX2 is selectively expressed in gastrointestinal adenocarcinomas.". Mod Pathol 17 (11): 1392-9. doi:10.1038/modpathol.3800205. PMID 15205684.
  21. Anatelli F, Chuang ST, Yang XJ, Wang HL. (2008). "Value of glypican 3 immunostaining in the diagnosis of hepatocellular carcinoma on needle biopsy". Am J Clin Pathol. 130 (2): 219-23?. doi:10.1309/WMB5PX57Y4P8QCTY. PMID 18628090.
  22. The diagnostic value of hepatocyte paraffin antibody 1 in differentiating hepatocellular neoplasms from nonhepatic tumors: a review. Lamps LW, Folpe AL. Adv Anat Pathol. 2003 Jan;10(1):39-43. Review. PMID 12502967.
  23. Lei JY, Bourne PA, diSant'Agnese PA, Huang J (April 2006). "Cytoplasmic staining of TTF-1 in the differential diagnosis of hepatocellular carcinoma vs cholangiocarcinoma and metastatic carcinoma of the liver". Am. J. Clin. Pathol. 125 (4): 519–25. doi:10.1309/59TN-EFAL-UL5W-J94M. PMID 16627262.
  24. 24.0 24.1 Marchevsky AM (March 2008). "Application of immunohistochemistry to the diagnosis of malignant mesothelioma". Arch. Pathol. Lab. Med. 132 (3): 397-401. PMID 18318582. http://journals.allenpress.com/jrnlserv/?request=get-abstract&issn=0003-9985&volume=132&page=397.
  25. Goldstein NS, Bassi D (May 2001). "Cytokeratins 7, 17, and 20 reactivity in pancreatic and ampulla of vater adenocarcinomas. Percentage of positivity and distribution is affected by the cut-point threshold". Am. J. Clin. Pathol. 115 (5): 695–702. doi:10.1309/1NCM-46QX-3B5T-7XHR. PMID 11345833.
  26. Geller SA, Dhall D, Alsabeh R (March 2008). "Application of immunohistochemistry to liver and gastrointestinal neoplasms: liver, stomach, colon, and pancreas". Arch. Pathol. Lab. Med. 132 (3): 490–9. PMID 18318589.
  27. Perry, Arie; Brat, Daniel J. (2010). Practical Surgical Neuropathology: A Diagnostic Approach: A Volume in the Pattern Recognition series (1st ed.). Churchill Livingstone. pp. 12. ISBN 978-0443069826.
  28. Pernick NL, DaSilva M, Gangi MD, Crissman J, Adsay V (November 1999). ""Histiocytic markers" in melanoma". Mod. Pathol. 12 (11): 1072–7. PMID 10574605.
  29. URL: http://www.ncbi.nlm.nih.gov/omim/605545. Accessed on: 3 February 2011.
  30. URL: http://www.abcam.com/Macrophage-antibody-MAC387-FITC-ab7429.html. Accessed on: 3 February 2011.
  31. URL: http://www.abcam.com/Macrophage-antibody-MAC387-ab49408.html. Accessed on: 3 February 2011.
  32. URL: http://www.ncbi.nlm.nih.gov/omim/604862. Accessed on: 2 February 2011.
  33. 33.0 33.1 Alkushi A, Irving J, Hsu F, et al. (March 2003). "Immunoprofile of cervical and endometrial adenocarcinomas using a tissue microarray". Virchows Arch. 442 (3): 271-7. doi:10.1007/s00428-002-0752-4. PMID 12647218.
  34. URL: http://www.nature.com/modpathol/journal/v19/n8/full/3800620a.html
  35. [Evaluation of immunohistochemical markers for differential diagnosis of hepatocellular carcinoma from intrahepatic cholangiocarcinoma] Dong H, Cong WL, Zhu ZZ, Wang B, Xian ZH, Yu H. Zhonghua Zhong Liu Za Zhi. 2008 Sep;30(9):702-5. Chinese. PMID 19173916.
  36. Nonaka, D.; Chiriboga, L.; Soslow, RA. (Oct 2008). "Expression of pax8 as a useful marker in distinguishing ovarian carcinomas from mammary carcinomas.". Am J Surg Pathol 32 (10): 1566-71. doi:10.1097/PAS.0b013e31816d71ad. PMID 18724243.
  37. Kanner, WA.; Galgano, MT.; Stoler, MH.; Mills, SE.; Atkins, KA. (Oct 2008). "Distinguishing breast carcinoma from Müllerian serous carcinoma with mammaglobin and mesothelin.". Int J Gynecol Pathol 27 (4): 491-5. doi:10.1097/PGP.0b013e31817d5340. PMID 18753974.
  38. URL: http://atlasgeneticsoncology.org/Genes/BCL1.html. Accessed on: 17 December 2010.
  39. Humphrey, Peter A; Dehner, Louis P; Pfeifer, John D (2008). The Washington Manual of Surgical Pathology (1st ed.). Lippincott Williams & Wilkins. pp. 567. ISBN 978-0781765275.
  40. Cheville JC, Rao S, Iczkowski KA, Lohse CM, Pankratz VS (April 2000). "Cytokeratin expression in seminoma of the human testis". Am. J. Clin. Pathol. 113 (4): 583–8. doi:10.1309/3QLC-5MF1-JYXU-A5XX. PMID 10761461.
  41. Tadrous, Paul.J. Diagnostic Criteria Handbook in Histopathology: A Surgical Pathology Vade Mecum (1st ed.). Wiley. pp. 107. ISBN 978-0470519035.
  42. URL: http://ajp.amjpathol.org/cgi/content/full/160/3/759. Accessed on: 3 August 2010.
  43. Hadzi-Mihailovic, M.; Cakic, S.; Jankovic, S.; Raybaud, H.; Nedeljkovic, N.; Jankovic, L.. "Ki-67 expression in oral lichen planus.". J BUON 17 (1): 132-7. PMID 22517707.
  44. http://www.genenames.org/data/hgnc_data.php?hgnc_id=21086
  45. Tazi, EM.; Lalya, I.; Tazi, MF.; Ahellal, Y.; M'rabti, H.; Errihani, H. (2010). "Transitional cell carcinoma of the ovary: a rare case and review of literature.". World J Surg Oncol 8: 98. doi:10.1186/1477-7819-8-98. PMID 21073751.
  46. 46.0 46.1 46.2 46.3 46.4 46.5 46.6 46.7 46.8 Bahrami, A.; Truong, LD.; Ro, JY. (Mar 2008). "Undifferentiated tumor: true identity by immunohistochemistry.". Arch Pathol Lab Med 132 (3): 326-48. doi:10.1043/1543-2165(2008)132[326:UTTIBI]2.0.CO;2. PMID 18318577.
  47. Llombart-Bosch A, Lopez-Guerrero JA, Peydro-Olaya A (2002). "Synovial sarcoma (SS): new perspectives supported by modern technology". Arkh. Patol. 64 (3): 39–47. PMID 15338724.
  48. Itakura E, Tamiya S, Morita K, et al. (September 2001). "Subcellular distribution of cytokeratin and vimentin in malignant rhabdoid tumor: three-dimensional imaging with confocal laser scanning microscopy and double immunofluorescence". Mod. Pathol. 14 (9): 854–61. doi:10.1038/modpathol.3880401. PMID 11557780. http://www.nature.com/modpathol/journal/v14/n9/full/3880401a.html.
  49. Miettinen M, Fanburg-Smith JC, Virolainen M, Shmookler BM, Fetsch JF (August 1999). "Epithelioid sarcoma: an immunohistochemical analysis of 112 classical and variant cases and a discussion of the differential diagnosis". Hum. Pathol. 30 (8): 934–42. PMID 10452506.
  50. URL: http://cat.inist.fr/?aModele=afficheN&cpsidt=2504165. Accessed on: 26 April 2011.

External links