[Histonet] RE: Secondary antibody causing nuclear staining

[Reynolds,Donna M]

If you are running a negative control (no primary)with your ABC staining wouldn't you see the same nuclear labeling in the NC ? Thus alerting you to false staining and indicating that you should try a HRP conjugated secondary or use a polymer system. 
Good discussion thank Tony. 
Donna Reynolds HT(ASCP) Chief Histologist IHC Lab

-----Original Message-----
> I understand the point about the biotin and I should have said that 
> when using the ABC method we have taken to always using an 
> avidin/biotin blocking kit. We are using biotinylated secondary 
> antibodies from Vector. I have seen the same problem occur in our 
> anti-mouse, anti-rabbit and anti-goat. In my last run I had stomach 
> fundus as well as skin melanoma, both had pos.nuclei in the negative 
> (no primary). In another run I had colon ca and breast ca, the breast 
> ca had fewer pos. nuclei than the colon ca but they were still there. 
> Some days the positive nuclei are stronger in a sample that was just 
> weakly positive before. Just want to understand what it is and what effects it.
> Thank you all for your ideas.
> Eva Permaul
> Georgetown University
> 
> On Mon, Jul 23, 2012 at 7:16 PM, Tony Henwood (SCHN) < 
> tony.henwood <@t> health.nsw.gov.au> wrote:
> 
>> 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
>>