slide to be stained for CA125 comprised:
1. Fallopian tube, 2. Appendix, 3. Colon adenocarcinoma, 4.
5. Ovarian serous carcinoma gr. 2, 6. Ovarian serous
carcinoma gr. 3.
All specimens were fixed in 10 % NBF.
Criteria for assessing a CA125 staining as optimal included:
A moderate to strong, predominantly membranous staining in the
epithelium of the fallopian tube.
A moderate to strong distinct predominantly membranous staining in
the majority of the neoplastic cells in the two ovarian serous
At least a focal membranous staining in the mesothelioma.
No staining of the appendix or the colon adenocarcinoma.
48 laboratories submitted stains. At the assessment 18 achieved
optimal marks (38 %), 14 good (29 %), 13 borderline (27 %) and 3 (6
%) poor marks.
The following Abs were used:
mAb clone OC125 (Dako, n=21; Ventana, n=4; Laborel, n=1)
mAb clone Ov185:1 (Novocastra, n=14)
mAb clone M11 (Dako, n=6; HistoCIS, n=1)
mAb clone TA347 (Zymed, n=1)
Optimal staining for CA125 in this assessment was obtained with
following mAbs: clone Ov185:1 (8 out of 14), clone M11 (6 out of 7)
and clone OC125 (4 out of 26).
All optimal protocols irrespective of the clone used were based on
Heat Induced Epitope Retrieval (HIER).
With clone Ov185:1 the following HIER buffers were used in the
optimal protocols: Tris-EDTA/EGTA pH 9 (5 out of 8 were optimal),
EDTA/EGTA pH 8 (1 out of 1 was optimal), Citrate pH 6 (1 out of 3
was optimal) and Target Retrieval Solution S1699 (Dako, 1 out of 1).
In the optimal protocols Ov185:1 was typically used in the range of
1:30 – 1:200 depending on the sensitivity of the protocol applied.
With clone M11, the following HIER buffers were used in the optimal
protocols: Citrate pH 6 (3 out of 3 were optimal) Tris-EDTA/EGTA pH
9 (2 out of 3 were optimal) and EDTA pH 9 (1 out of 1 was optimal).
In the optimal protocols M11 was typically used in the range of 1:40
– 1:100 depending on the sensitivity of the protocol applied.
Alternatively a ready-to-use (RTU) Ab was used.
With clone OC125 the following HIER buffers were used in the optimal
protocols: Tris-EDTA/EGTA pH 9 (3 out of 10 were optimal) and Target
Retrieval Solution S1699 (Dako, 1 out of 1 was optimal). In the
optimal protocols OC125 was typically used in the range of 1:50 –
1:100 depending on the sensitivity of the protocol applied. With
clone OC125 other HIER buffers such as CC1 (n=6) or citrate pH 6
(n=5) could not be used to obtain an optimal result in this
assessment. Using CC1 all 6 laboratories had an insufficient
staining. However, CC1 was frequently combined with OC125 as an RTU
Ab (n=3). 3 out of 5 laboratories using citrate pH 6 had a good (but
not optimal) staining while 2 had an insufficient staining, of which
1 used OC125 as an RTU Ab.
The most frequent causes of insufficient staining were:
- Too low concentration of the primary Ab
- Using clone OC125 as a RTU Ab (3 out of 4 were insufficient)
- Less succesful primary Ab
- Inappropriate epitope retrieval (HIER in CC1 or citrate pH 6)
The prevalent feature of an insufficient staining was a too weak
staining of the epithelium in the fallopian tube and a false
negative staining of the mesothelioma. In general the majority of
the laboratories were able to detect CA125 in both ovarian serous
adenocarcinomas. In the optimal protocols the membranes of both the
normal epithelial cells of the fallobian tube and the membranes of
the neoplastic cells in the mesothelioma distinctively were
demonstrated. In the insufficient staining the epithelium of the
fallopian tube was weakly labelled and the mesothelioma was
negative. In general the mAb clone M11 labelled a higher proportion
of both normal mesothelial cells and the neoplastic mesothelial
cells compared to the clones Ov185:1 and OC125.
MAb clone M11 (Dako, HistoCIS)
seems to be the most sensitive marker for CA125.
HIER seems to be the most appropriate pre-treatment for the clones
Ov185:1, M11 and OC125.
The epithelium in the fallopian tube is a reliable positive control.
The staining reaction should be strong, membranous.