slide to be stained for
Malignant mesothelioma, epithelial type, 4.
adenocarcinoma, high grade, 5. Colon adenocarcinoma,
Criteria for assessing a CEA
staining as optimal included: A moderate to strong, distinct
cytoplasmic and membranous staining of enterocytes of the
appendiceal mucosa and the superficial squamous epithelial cells of
the oesophagus, as well s a strong, distinct cytoplasmic and
membranous staining of the two colon adenocarcinomas. Staining
reaction should not be seen in any other cells including neutrophils,
histiocytes (with the exception of those in the vicinity of CEA-positive
tumour cells), liver cells (bile canaliculi)
or the malignant
If a slight staining reaction in neutrophils and histiocytes was seen in an
otherwise optimal staining, it was accepted as ‘good’. In case of
strong reaction in neutrophils and histiocytes as well as staining
of bile canaliculi (as demonstrated with all polyclonal antibodies [pAbs]
and a few monoclonals [mAbs]), the antibody was considered
inappropriate, because of its capability of cross reacting with
75 laboratories submitted
stains. Of these 15 used a CEA antibody considered inappropriate.
Assessing the remaining 60 stains, 37 were found optimal (61 %), 15
good (25 %), 7 borderline (12 %) and 1 (2 %) poor.
The following appropriate
mAbs were used:
Clone II-7 from DakoCytomation (56)
Clone Col-1 from Zymed (n=3)
Clone BW 431/26 from Behring (n=1).
In this assessment optimal
staining could be obtained both with clone II-7 (36 out of 56 (64
%)) and clone Col-1. (1 out of 3).
To obtain an optimal staining
with clone II-7, all used HIER with either Tris-EDTA/EGTA pH 9 as
the heating buffer, (29 out of 36 (81%) were optimal), Citrate pH 6 – 7,3 (4 out
of 10 (40%) were optimal), TRS pH 6 (DakoCytomation)(3 out of 3
were optimal), or CC1 (Ventana
Benchmark)(1 out of 2 was optimal). None of the 4 laboratories using proteolytic pre-treatment obtained an optimal staining.
The mAb clone II-7 was typically used in a dilution of 1:50 –
The optimal protocol for the mAb clone Col-1 was based on HIER using
Tris-EDTA/EGTA pH 9 as the heating buffer with a primary antibody
dilution of 1:100.
The insufficient stains typically displayed a positive cytoplasmic reaction of the high
grade colon adenocarcinoma, whereas the membranous reaction of the
low grade colon adenocarcinoma generally was too weak or totally
When excluding the inappropriate stains, the
most frequent causes of insufficient staining were:
- Inappropriate epitope retrieval (proteolytic
- Omission of epitope retrieval
- Too low concentration of the primary antibody.
The following mAbs were
for the above mentioned
12-140-10, clone B01-94-11M, clone C260, clone TF-3H8-1, and clone
Using an inappropriate CEA-antibody, neutrophils, histiocytes and sometimes bile canaliculi were stained,
heterogeneous staining reaction of the malignant mesothelioma was observed as well.