[Histonet] Secondary antibody causing nuclear staining?

Tue Jul 24 14:32:58 CDT 2012

Yes. The strength of the stained nuclei in the no primary slides are
stronger on some days than others.

On Tue, Jul 24, 2012 at 8:18 AM, Kim Donadio <one_angel_secret <@t> yahoo.com>wrote:

> Are you getting false positives and variations on the same control tissue
> for different days ?
>
> Sent from my iPhone
>
> 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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> >>
> >>
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