[Histonet] Secondary antibody causing nuclear staining?

Tue Jul 24 07:18:48 CDT 2012

Are you getting false positives and variations on the same control tissue for different days ? 

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On Jul 24, 2012, at 8:13 AM, Eva Permaul <eca9 <@t> georgetown.edu> wrote:

> 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
>> 
>> -----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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