slide to be
stained for MLH1
comprised: 1: Appendix, 2: Colon adenocarcinoma with loss of MLH1,
3: Colon adenocarcinoma with loss of MSH2.
All tissues were fixed in 10% neutral buffered formalin.
assessing an MLH1 staining as optimal included: A strong and
nuclear staining of virtually all benign cells (enterocytes, lymphocytes,
smooth muscle cells etc.) in the appendix and the two colon
specimens. The neoplastic cells of the adenocarcinoma with loss of
MLH1 (specimen 2) should be negative, while the neoplastic cells of
adenocarcinoma with loss of MSH2 should be positive (specimen 3). A weak cytoplasmic
reaction was accepted.
laboratories participated in the assessment. At the assessment 8 achieved optimal (32 %), 10
good (40 %), 5 borderline (20 %) and 2 (8 %) poor marks.
antibody clones were used:
mAb clone G168-15 (BD Pharmingen, n=20; Biocare, n=2)
mAb clone 168-728 (Cell Margue, n=3).
assessment optimal staining could only be obtained with the mAb
clone G168-15 (8 out of 22 were optimal). The optimal staining was
based on HIER in all cases with heating buffers and devices as
follows: Tris-EDTA/EGTA pH 9 (6 out of 13 were optimal), EDTA pH 8 (1 out of 4) or
Borg Decloaker pH 9,5 (Biocare) (1 out of 1); MWO (5 out of 18 were
optimal) or pressure
cooker (3 out of 4). Clone G168-15 was typically used in dilution of of
1:25 – 1:200.
enterocytes and germinal centre cells were demonstrated in most
protocols, but in the optimal staining all enterocytes, lymphocytes,
stromal cells etc. were labelled, the latter are always are found
included in the neoplastic lesions as an internal control. The colon
adenocarcinoma with the MLH1 loss displayed a slight variation in
the staining characteristics: in some stains the neoplastic cells
were totally negative while in other stains, a
nuclear membranes was observed in some cells.
This may be the result
of a highly sensitive protocol (or slight over staining) and should
not be interpreted as positive.
frequent causes of insufficient staining were:
- Too diluted primary antibody
- Insufficient HIER.
feature of an insufficient staining was a too weak or negative
staining of the majority of the cells that were expected to stain.
As the identification of an MLH1 loss is characterized by a negative
immunoreaction of the neoplastic cells, it is of decisive importance
that the benign cells can be demonstrated and thus serves as
internal positive control.