[Histonet] Secondary antibody causing nuclear staining?

[Tony Henwood (SCHN)]

I should have added that this was from the workshop notes on a Hypotheticals Workshop I ran last year at our Australian National Meeting.

Regards 
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) 
Laboratory Manager & Senior Scientist 
Tel: 612 9845 3306 
Fax: 612 9845 3318 
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA 


-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Tony Henwood (SCHN)
Sent: Tuesday, 24 July 2012 9:00 AM
To: 'Eva Permaul'; histonet <@t> lists.utsouthwestern.edu
Subject: RE: [Histonet] Secondary antibody causing nuclear staining?

It is possible that this is due to "Biotin nuclei" where excess biotin is found in the nuclei of some cells, see below:

Optically clear nuclei have been reported in endometrial epithelium associated with first and second trimester abortions (Sickel & di Sant'Agnese 1994). Optically clear nuclei have also been found in different types of tissues of diverse organs such as ovary, thyroid and lung (Nakatani et al 1994, Mount & Cooper 2001). The optically clear nuclei contain excess biotin.

Endogenous biotin immunoreactivity is generally not visualized in formalin fixed, paraffin-embedded tissues unless a heat-induced antigen retrieval step has been introduced (Mount & Cooper 2001).

In this placental section, optically clear nuclei (containing biotin) bind to the streptavidin of the ABC technique giving a reaction similar to that seen with CMV containing cells. If a polymer method (or even the original Sternberger's PAP method) is used then this anomalous staining will disappear, thus allowing confident demonstration of CMV infected nuclei.

The false-positive staining pattern caused by endogenous biotin can be cytoplasmic or nuclear. A report of positive immunoreactivity of hepatocellular carcinomas for inhibin was later determined to be a false-positive finding due to cytoplasmic endogenous biotin. Steroid cell tumours of the ovary were found to demonstrate endogenous biotin cytoplasmic staining in 36% of cases. Immunoreactivity for anti-Herpes virus immunohistochemical staining in a series of endometria was also later determined to be a false-positive result due to biotin. The prominent intranuclear inclusions, resembling herpes virus cytopathic effect, were caused by intranuclear biotin and not viral particles. Similar false positive staining for CMV in products of conception has also been reported (Mount & Cooper 2001). 

False-positive staining can be cytoplasmic or nuclear. When cytoplasmic, the appearance of the false signal is that of a dull brown granular or fluffy staining pattern. If this quality of staining is observed with several different antibodies, endogenous staining by biotin should be considered. When nuclear, a false-positive reaction may be associated with optically clear nuclei identified on H&E stained sections. False-positive staining due to endogenous biotin, however, does not occur in a cell membrane pattern (Mount & Cooper 2001).

Mount SL & Cooper K (2001) "Beware of biotin: a source of false-positive immunohistochemistry" Current Diagnostic Pathology  7:161-167.
Nakatani et al (1994) Am J Surg Pathol 18(6):637-642.
Sickel & di Sant'Agnese (1994) Arch Pathol Lab Med 118:831-833


Regards
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) Laboratory Manager & Senior Scientist
Tel: 612 9845 3306
Fax: 612 9845 3318
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead Locked Bag 4001, Westmead NSW 2145, AUSTRALIA 

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Eva Permaul
Sent: Monday, 23 July 2012 11:40 PM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] Secondary antibody causing nuclear staining?

Hello,

I have noticed that our biotinylated secondary antibodies on occasion cause nuclear staining in some samples. Why is this? It is not every time so I find it rather stange. Anyone know why this is happening and what I can do to avoid it?

Thank you for any suggestion,
Eva Permaul
Georgetown University
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