I do not have an answer for any of Vinnie's questions except to say that I have experienced the same thing and I would be interested in any feedback that is generated.

Martha Ward
Wake Forest University Baptist Medical Center

-----Original Message-----
From: Vinnie Della Speranza [mailto:dellav@musc.edu] 
Sent: Monday, March 31, 2003 4:45 PM
To: histonet@pathology.swmed.edu
Subject: Mart-1 IHC- I need your expertise please!!


I'd appreciate any insight anyone may offer with the following.

we frequently perform Mart-1 ihc staining to demonstrate melanocytes. We use a monoclonal antibody from Dako (clone A103 working dilution
1:50)  for this marker and an alkaline phosphatase detection kit from Biogenex utilizing a red chromogen. we perform high temperature antigen retrieval in citrate buffer pH 6.0

our positive control is a nevus selected by one of our dermatopathologists. we generally prepare a working dilution of this antibody once each week

from time to time, we've received feedback that "not enough melanocytes are staining" which causes the dermpathologist to conclude that the antibody is not performing to expectations. Slides are always checked prior to being turned in to verify that we have achieved the expected pattern of staining, however we do not attempt to quantitate the # of slides appearing positively stained with this marker.

I reviewed a case today that had been rejected by the pathologist. In evaluating the epidermis of four pieces of excised skin (FFPE), I found evidence of appropriate staining in each, although some areas of the epidermis contained many positive cells, some areas only a few, some areas none at all. the melanocytes in our control nevus stained with the expected pattern and intensity.

My questions to you are as follows:

has anyone found variable Mart-1 staining in tissues such as I've described and if so, can you elaborate as to it's suspected cause?

is it possible that handling of the specimen prior to fixation and processing could cause the result we are experiencing?

is it possible that pretreatment of the surgical site in the patient, including the use of topical or injected anesthetics or skin disinfectants could cause variable Mart-1 staining?

is there something about the stability of this antibody that we haven't considered that may be contributing to this situation?

is it possible that the pathologist expected a different outcome and thus concluded that the stain results were incorrect? your thoughts are most appreciated.






Vinnie Della Speranza
Manager for Anatomic Pathology Services
Medical University of South Carolina
165 Ashley Avenue  Suite 309
Charleston, SC 29425
Ph: 843-792-6353
fax: 843-792-8974