[Histonet] RE: Secondary antibody causing nuclear staining
Reynolds,Donna M
dreynold <@t> mdanderson.org
Wed Jul 25 09:04:46 CDT 2012
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
>>
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