slide to be stained for Pan-cytokeratin (pan-CK) comprised:
1. Appendix, 2. Liver, 3. Tonsil, 4. Breast lobular carcinoma, 5.
Criteria for assessing a CK-pan staining as optimal included:
A strong and distinct cytoplasmic reaction in the appendix
enterocytes (including crypt basis) and bile ducts, and a moderate
predominantly membranous reaction of the large majority of
A strong and distinct cytoplasmic reaction of the squamous
epithelial cells in the tonsil and at least a faint staining of
fibroblastic reticulum cells in the lymphoid tissue of the tonsil.
A strong and distinct cytoplasmic reaction of the breast lobular
Focally, a strong and distinct cytoplasmic reaction of the
85 laboratories submitted stains. At the assessment 21 achieved
optimal marks (25 %), 28 good (33 %), 29 borderline (34 %) and 7 (8
%) poor marks.
The following Abs were used:
mAb clone cocktail 5D3 + LP34 (Novocastra, n=3)
mAb clone Ab-2 (NeoMarkers, n=2)
mAb clone cocktail AE1/AE3 (Boehringer, n=1; Chemicon, n=1; Dako, n=43;
NeoMarkers, n=1; Zymed, n=3)
mAb clone cocktail AE1/AE3 + 5D3 (Biocare, n=3)
mAb clone cocktail AE1/AE3 + PCK26 (Ventana, n=7)
mAb clone KL1 (Immunotech n=9, Serotec n=2)
mAb clone MNF116 (Dako,
mAb clone Lu-5 (NeoMarkers, n=2)
Furthermore, two laboratories used mAb clone CAM5.2 (Becton Dickinson)
which is considered inappropriate as a pan-CK marker as it only
detects low molecular weight CKs.
Optimal staining for pan-CK in this assessment was obtained with the
following mAb clones/clone cocktails: Ab-2 (2 out of 2), AE1/AE3 (13 out of 48)
AE1/AE3 + 5D3 (2 out of 3), KL1 (2 out of 11) and MNF116 (3 out of
8). However, for the other clones, we consider it likely that
optimal results could have been obtained using other protocols
(e.g., using HIER instead of proteolytic epitope retrieval).
However, we have not yet tested this in our own laboratories.
All optimal protocols for the above mentioned clones were based on
HIER except for the clone MNF116, for which the optimal protocols
were based on proteolytic pre-treatment.
In the optimal protocols using the mAb clone Ab-2 Tris-EDTA/EGTA pH
9 was used as HIER buffer (2 out of 2), with the mAb used in the
range of 1:300 – 1:400 depending on the sensitivity of the protocol
In the optimal protocols using the mAb clone AE1/AE3 both citrate pH
6,0, CC1 (Ventana), and Tris-EDTA/EGTA pH 9 could be used as the
HIER buffer (1 out of 5, 1 out of 2, and 10 out of 22 were optimal,
respectively), with the mAb used in the range of 1:20-1:250
depending on the sensitivity of the protocol applied.
In the optimal protocols using the mAb clone AE1/AE3 + 5D3
Tris-EDTA/EGTA pH 9 was used as the HIER buffer (2 out of 2) with
the mAb used in the range of 1:400-1:800 depending on the
sensitivity of the protocol applied.
In the optimal protocols using the mAb clone KL1 Tris-EDTA/EGTA pH 9
was used as HIER buffer (2 out of 9) with the mAb used in the range
of 1:50 – 1:75 depending on the sensitivity of the protocol applied.
In the optimal protocols using the mAb clone MNF116 the protocol was
based on proteolytic pre-treatment using Proteinase K and a dilution
of the mAb in the range of 1:50 – 1:400 depending on the sensitivity
of the protocol applied.
The most frequent causes of insufficient stainings were (often in
- Inappropriate choice of primary Ab
- Too low concentration of the primary antibody
- Inappropriate epitope retrieval (proteolysis for the mAb clone
The prevalent feature of an insufficient staining was a too weak or
negative reaction of cells/structures supposed to be demonstrated.
The majority of the laboratories (95 %) were capable to demonstrate
CK in the enterocytes of the appendix and the bile ducts of the
liver as well as the lobular breast carcinoma. However,
demonstration of CK in the seminoma was more difficult and only
demonstrated in approximately 60 % of the laboratories. All
laboratories demonstrating CK in the seminoma were capable of
detecting the relatively low expression of CK (CK type 8 and
hepatocytes, indicating that liver may be a reliable control
for the demonstration of low molecular weight CKs, provided that the
protocol is calibrated to stain the liver cells. In this
assessment all laboratories using the most widely used mAb clone
AE1/AE3 (n=59) either alone or in cocktail with PCK26 basing the
protocol on proteolytic pre-treatment (n=12) had an insufficient
staining due to a negative staining reaction of the hepatocytes.
Simultaneously with the demonstration of low molecular weight CK a
pan-CK Ab should also identify the high molecular weight CK in
squamous epithelial cells. In this assessment only few laboratories
had problems with the detection of CK in the squamous epithelial
cells of the tonsil. The reason for insufficient demonstration of
high molecular weight was typically due to an inappropriate Ab (as
the mAb clone CAM5.2 which is a marker for low molecular weight CK
subtypes only) or a too low concentration of the Ab, especially when
using the clone KL1, which reacts with a more limited number of the
high molecular weight CK subtypes.
Pan-CK was also assessed in
run 8. In that run 72 laboratories
participated out of which 47 % (34 laboratories) was marked
insufficient. Each laboratory was given a specific recommendation to
improve their protocol. 29 laboratories, which obtained an
insufficient result in run 8, submitted a new pan-CK stain in run
15. 18 of the laboratories followed the recommendation and 13
improved the result from insufficient to either good or optimal (72
%). 11 laboratories did not follow the recommendation and 1 of these
(9 %) obtained a sufficient staining in run 15.
However, the overall proportion of insufficient staining was in this
run only reduced to 42 % from 47 % in run 8, and as the proportion
of insufficient staining still is relative high pan-CK will be
repeated in 2006 or 2007.
The mAb clones Ab-2, AE1/AE3, AE1/AE3 + 5D3, and
KL1 was found to be
the most successful markers for pan-CK using HIER. The mAb clone
found to be useful for pan-CK using proteolytic
pre-treatment. Liver and tonsil in combination are appropriate
control tissues. The large majority of hepatocytes should be