RE: [Histonet] MLH-1

From:"Young Kwun"

Dear Mary,

I would like to make a point about the antibody you are using which is
Zymed's MLH-1 (clone 14).

As you know, MLH1 antibody is one of the most important colon cancer
markers. It is particularly useful in determining microsatellite instability
(MSI) status and tumors arising in patients with hereditary non-polyposis
colorectal cancer (HNPCC) syndrome.

MLH1 is notorious for its capricious staining manner in some cases and I
tried several MLH1 antibodies on the market before I decided to use BD
Pharmingen's (clone G168-15) for last 5 years. Occasionally I still had some
problem with the stain so I decided to try Zymed's MLH1 (clone 14) last
year. The staining was sometimes stronger than our routine BD's MLH1 so I
decided to use Zymed's MLH1 (clone 14) as our routine one during the year
2005. With the help of some new and more sensitive detection system recently
(two step polymer instead of one step), I was able to produce good stains
for MSI test. Our panel also includes MHS2, MSH6, PMS2 and MGMT. It is well
known that MLH1 delete and PMS2 delete are occurring together and it is very
rare for a case showing only PMS2 negative. During the last year, we had
several cases of colon cancer which appeared to be PMS2 delete only. I had
to repeat MLH1 stains to confirm the result in some cases. 

When I compared the two MLH1 clones separately, the result was so dramatic.
Our old clone (BD Pharmingen G168-15) showed a clean MLH1 delete in tumour
cells only, whereas clone 14 MLH1 showed a strong positive staining in the
tumour cells as well. Both clones showed a strong positivity in the normal
epithelial cells. Clone 14 MLH1 clearly demonstrated false positive
Therefore I had to stop using the clone14 MLH1 immediately. I used 1/100
dilution and if I dilute 1/300 or 1/500 the normal epithelium would not
stain. I also tried different Lot number and again showed false positivity.

So I had to repeat all the colon carcinoma cases during the year 2005 and
found that many MLH1 (and PMS2) delete cases were reported incorrectly. In
fact I would like to know that whether you've ever seen a delete case with
the clone 14 MLH1.
These results were also confirmed by MSI molecular biological test at St.
Vincent Hospital here in Sydney.

PS: We are now using BondMax autostainer for MSI test and I don't need to
incubate overnight anymore. The staining is fantastic!

Young Kwun
Senior Hospital Scientist
Dept of Anatomical Pathology
Concord Hospital
Concord NSW 2139
Phone:61-2-9767 6075
Fax:61-2-9767 8427


-----Original Message-----
[] On Behalf Of Mary Judd
Sent: Wednesday, 21 June 2006 8:31 PM
Subject: [Histonet] MLH-1

We use MLH-1(14) from Zymed 18-2342
We pressure cook in citrate buffer 6pH, use the antibody at 1:30 and
incubate overnight at 4C.

Mary Judd
Section Head Immunohistochemistry
Cellular Pathology
Level E, Mailpoint 2
Southampton University Hospitals Trust
Tel. 02380 795144
Fax. 02380 796869

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