Abcam Ad

Author Topic: urgent : melanA/HMB45 FALSE POSITIVE?  (Read 13010 times)

0 Members and 1 Guest are viewing this topic.

Offline philtrougouboff

  • SuperTech
  • ****
  • Posts: 22
urgent : melanA/HMB45 FALSE POSITIVE?
« on: August 03, 2003, 08:10:34 AM »
:?
Does anybody knows about the possibility of MelanA/Hmb45 staining any other cell than metastatic melanocytes in a lymph node  :?:  (e.g. reticulum cells or histiocytes or....)
Urgent because I have to resolve the problem for a bilateral axillary adenopathy excised in a woman with a 2mm deep melanoma of the back.
Thanks for the help. :D
Phil
hilippe Trougouboff, MD

urgent : melanA/HMB45 FALSE POSITIVE?
« on: August 03, 2003, 08:10:34 AM »

Offline richard03

  • Administrator
  • GoldMember
  • *****
  • Posts: 1212
urgent : melanA/HMB45 FALSE POSITIVE?
« Reply #1 on: August 06, 2003, 03:04:42 PM »
Do you have both positive and negative controls. These controls should be always included in the immunostaining. You may also need antibody controls (such as specificity test) or different markers such as S100/tyrosinase, etc.

Richard

Offline philtrougouboff

  • SuperTech
  • ****
  • Posts: 22
MelanA/HMB45
« Reply #2 on: August 07, 2003, 02:00:22 AM »
:D
Hi Richard, thanks for your answer. The Ab we use is a mixture of 2 clones of MelanA and one of Hmb45. We do use positive control. Meanwhile I also made a negative one that shows the same positive cells. It seems that they are some kind of reticulum/dendritic cells. This "false" positive cells are not mentioned in the data sheet of the Ab  :? .I don't know if the staining is due to real binding of the Ab or something non-specific. It could be worrysome when you are dealing with Sentinel Lymph Node and searching for micrometastases. If I'd perform a S100 stain I know that there is S100 +ve cells in a reactive LN, so we don't use it for searching there melanoma cells.
Phil
hilippe Trougouboff, MD

Offline richard03

  • Administrator
  • GoldMember
  • *****
  • Posts: 1212
urgent : melanA/HMB45 FALSE POSITIVE?
« Reply #3 on: August 07, 2003, 10:32:55 AM »
Based on the info you provided that the Negative Control showed same positive staining, I would consider the problem came from either secondary antibody or endogenous peroxidase/biotin (if you used ABC method). You may need more blocking steps.

I am curious about why you mix those three primary antibodies. Is there a potential problem in creating nonspecific staining or you may be doing triple labeling?

Lastly, if you want to confirm if this is real binding, you may consider to do a absorption control for this antibodies.

Richard

Offline philtrougouboff

  • SuperTech
  • ****
  • Posts: 22
urgent : melanA/HMB45 FALSE POSITIVE?
« Reply #4 on: August 08, 2003, 02:30:17 AM »
Hi Richard,
The Ab we use is from Biocare Medical, it is a concentrated monoclonal Ab cocktail mixing three clones. The use of a "cockail" allowed a better sensitivity than each one alone. The "ready to use" mix is cheaper than using two or three Ab separately.
I think that the non speciifc positivity is probably endogenous biotin activity, as you suggest. We are using ABC tech for his stain, with pretreatment using Biocare's Nucleardecloaker (high pH buffer) according to the recommendation of the manufacturer. Could it be a problem with the pretreatment when there is a problem of non-specific stain in general :?:  
Thanks for you interest,
Phil
hilippe Trougouboff, MD

Offline richard03

  • Administrator
  • GoldMember
  • *****
  • Posts: 1212
urgent : melanA/HMB45 FALSE POSITIVE?
« Reply #5 on: August 08, 2003, 04:07:57 PM »
Phil,

Yes. In general, heat induced antigen retrieval using buffer solutions will bring up endogenous biotin activity. So that is probably the reason for the nonspecific staining you got. But the "cocktail' makes me worried too. When sensitivity rose, specificity reduced. Anyway, try biotin block and see how it goes.

Richard

Offline philtrougouboff

  • SuperTech
  • ****
  • Posts: 22
melan/hmb45
« Reply #6 on: August 10, 2003, 02:27:52 AM »
Thanks a lot Richard, for your very useful tips  :idea: . It's important to know it for many stains. I'll try to do more strong biotin blocker as you suggest.
Do you know some specific blocker that is especially efficient?
Best wishes.

Phil
hilippe Trougouboff, MD

Offline ole

  • SilverMember
  • *****
  • Posts: 88
Re: melan/hmb45
« Reply #7 on: August 11, 2003, 03:58:08 AM »
Quote from: "philtrougouboff"
Thanks a lot Richard, for your very useful tips  :idea: . It's important to know it for many stains. I'll try to do more strong biotin blocker as you suggest.
Do you know some specific blocker that is especially efficient?
Best wishes.

Phil


Hi
I think this nonspesific staining might be caused by contamination of the primary antibody solution (as Richard mentioned) (the cocktail might be the problem).  I dont think blocking steps helps in this case. However VECTOR Avidin/Biotin blocking kit (SP-2001) is a good blocking kit.
I think you should try the melanoma markers seperately.
Take a look at WWW.nordiqc.org/. Under protocols are some useful info about melan-a and hmb45.

Ole

Re: melan/hmb45
« Reply #7 on: August 11, 2003, 03:58:08 AM »