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.henw...@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-boun...@lists.utsouthwestern.edu [mailto: >> histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Tony Henwood >> (SCHN) >> Sent: Tuesday, 24 July 2012 9:00 AM >> To: 'Eva Permaul'; histonet@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 >> _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet