slide to be stained for
Breast ductal carcinoma, 2.
Malignant epithelial mesothelioma, 4. Lung
adenocarcinoma, 5. Urothelial carcinoma.
Criteria for assessing a
staining as optimal included: A strong and distinct cytoplasmic
staining of the squamous epithelial cells of the tonsil,
myoepithelial cells lining breast glands and ducts (with normal
secretory epithelium or ductal carcinoma in situ), malignant
mesothelioma and urothelial carcinoma, whereas no reaction should be
seen in the breast in situ and invasive ductal carcinoma or lung
submitted stainings. One lab used an inappropriate Pan-CK ab. At the
assessment of the 73 stains, 34 achieved optimal (47 %), 22 good (30
%), 14 borderline (19 %) and 3 (4 %) poor result.
The following mAbs was
Producer, number of stains
||DakoCytomation, n=34; Zymed, n=3; Chemicon, n=2;
Cell Marque, n=1
||CK 1,5,10,14, and unidentified CK
||DakoCytomation, n=12; Ventana, n=3; Cell Marque
n=1; Enzo, n=1
In this assessment optimal staining could be obtained with the
mAbs clone XM26 (12/16 = 75% using this clone), mAb clone D5/16 B4
(22/40 = 55% using this clone) and 34BE12 (1/17 = 6% using this
In the optimal protocols with clone
XM-26, all used HIER with the following heating buffers: Tris-EDTA/EGTA
pH 9 (7 out of 7 using this buffer had an optimal result), CC1 (Ventana;
2 out of 2 using this buffer), TRS High pH 9,9 (DakoCytomation; 1
out of 1 using this buffer) and Citrate pH 6 (2 out of 7 using this
buffer). mAb XM26 was typically used in the range of 1:25 – 150.
In the optimal protocols with clone D5/16 B4, all of 22 laboratories
used Tris-EDTA/EGTA pH 9 as the heating buffer. D5/16 B4 was
typically used in the range of 1:25 – 200.
The optimal protocol for 34BE12 was based on
proteolytic pretreatment using
Protease 2 (Ventana) combined with a prediluted primary Ab. In otherwise optimal stains,
when HIER was used, clone 34BE12 gave a staining of breast secretory cells and ductal carcinoma in
situ (which were negative when the CK5 og CK5/6 Abs were used),
hampering the use of 34BE12 for the identification of CK5 and
myoepithelial cells. It should be emphazised that this cross
reaction is not seen in the prostate (sections not included
in this run).
The majority of the
laboratories were capable of detecting CK5 in the tonsil squamous
epithelium, the malignant mesothelioma and urothelial carcinoma,
whereas the insufficient staining typically revealed a too weak or false negative
staining in the breast myoepithelial cells.
most frequent causes of insufficient staining were:
- Omission of epitope retrieval
- Too low concentration of the primary Ab.
- False positive staining due to endogenous biotin.
Fig. 3a. Optimal CK5/6 staining of the breast ductal carcinoma.
A strong cytoplasmic staining is seen in the myoepithelial cells, with no
staining of the carcinoma in situ and the invasive carcinoma (upper
part of the photo). Same protocol as in Fig. 1a.
Fig. 3b. Insufficient CK5/6 staining of the breast ductal carcinoma
(compare with Fig. 3a, same field).
The myoepithelial cells are virtually unstained. Same protocol as in Fig. 1b and 2b.
Fig. 3c. Left: Clone 34BE12 staining of the breast ductal carcinoma using HIER.
A strong cytoplasmic staining is seen in the CIS.
Right: Clone 34BE12 staining of the breast ductal
carcinoma using proteolytic pretreatment. Staining is seen in the myoepithelial cells,
whereas no staining is seen in the CIS.