• A strong, distinct cytoplasmic reaction of virtually all the bile ductal epithelial cells, and at least a moderate, predominantly membranous reaction of the large majority of hepatocytes.
• A strong, distinct cytoplasmic reaction of virtually all the breast ductal epithelial cells.
• A moderate to strong, distinct staining of the majority of the neoplastic cells of the renal cell carcinoma, the small cell lung carcinoma and the colon adenocarcinoma.
• No staining of the esophageal squamous epithelial cells, except for a staining of the basal cells, when using an Ab reacting with CK8.

108 laboratories submitted stains but 9 laboratories used an inappropriate antibody such as pan-CK or CK19. Among the 99 laboratories, 66 % achieved a sufficient mark. The results are summarized in Table 1.

Table 1. Abs and scores for CK-LMW, run 25

1) Proportion of sufficient stains (optimal or good), 2) Proportion of sufficient stains with optimal protocol settings only, see below.
* Claimed by Zymed to be CK8.

Following central protocol parameters were used to obtain an optimal staining:

Concentrated Abs
mAb clone DC10: The protocols giving an optimal result were all based on heat induced epitope retrieval (HIER) using Tris-EDTA/EGTA pH 9 (15/19)*, Cell Conditioning 1 (BenchMark, Ventana) (5/6), Target Retrieval Solution pH 9 (EnVision FLEX TRS high pH, Dako, (3/6), Bond Epitope Retrieval Solution 2 (Bond, Leica) (2/3) or Citrate pH 6 (1/1) as retrieval buffer. The mAb was typically diluted in the range of 1:20 – 1:200 depending on the total sensitivity of the protocol employed. Using these protocol settings 33 out of 34 (97 %) laboratories produced a sufficient staining (optimal or good).
* (number of optimal results/number of laboratories using this buffer)

mAb clone C51: The protocols giving an optimal result were all based on HIER using Tris-EDTA/EGTA pH 9 (4/4) or Target Retrieval Solution pH 9 (EnVision FLEX TRS high pH, Dako (2/3). The mAb was typically diluted in the range of 1:50 – 1:100 depending on the total sensitivity of the protocol employed. Using these protocol settings 6 out of 7 (86%) laboratories produced a sufficient staining (optimal or good).

mAb clone 5D3: The protocols giving an optimal result were all based on HIER using Tris-EDTA/EGTA pH 9 (3/7), Bond Epitope Retrieval Solution 2 (Bond, Leica) (2/2) or Citrate pH 6 (1/3) as retrieval buffer. The mAb was typically diluted in the range of 1:20 – 1:150 depending on the total sensitivity of the protocol employed. Using these protocol settings 9 out of 11 (82%) laboratories produced a sufficient staining.

mAb clone CAM 5.2: The protocol giving an optimal result was based on enzyme pre-treatment with Protease 1 (BenchMark, Ventana) (1/5) and the Ab was used undiluted.

Ready-To-Use Abs
mAb clone DC10, IR618, Dako: The protocol giving an optimal result was based on HIER using Target Retrieval Solution pH 9 (EnVision FLEX TRS high pH) and an incubation time in 20 min for the primary Ab and EnVision Flex as the detection system.

The most frequent causes of insufficient staining were:
- Less successful antibodies (e.g., 12/12 protocols based on the mAb clone 35BH11 gave an insufficient result)
- Inappropriate epitope retrieval (e.g. enzymatic pre-treatment for the mAb clone 5D3)
- Too low concentration of the primary Ab.

In this assessment and in concordance with the previous CK-LMW assessments (run 16 and 20) the prevalent feature of an insufficient staining was a too weak or negative reaction of cells expected to stain. The majority of the laboratories were able to demonstrate CK-LMW in the bile duct epithelium and the colon adenocarcinoma. However, the demonstration of CK-LMW in the small cell lung carcinoma and especially the renal cell carcinoma was more difficult and only seen with appropriate protocol settings, e.g., a correct titre of the mAb clones DC10 and 5D3 combined with efficient HIER.
As observed in the previous assessments of CK-LMW, liver was a reliable positive control, as all laboratories that could demonstrate the membranous reaction in the hepatocytes also could demonstrate CK-LMW in the renal cell carcinoma, which in this run was the most challenging tumour.
The choice of Ab has a high impact on the pass rate, as e.g. the proportion of sufficient stains based on the mAb clone 5D3 was 72 % compared to 0 % ,when the mAb clone 35BH11 was used, despite the number of participants and otherwise applied protocol settings were similar for the two clones. In Table 2, the overall pass rates are summarized for the most widely used clones in the last three CK-LMW assessments.

Table 2: Performance of five commonly used clones in three runs

These data clearly indicates that the mAb clones CAM5.2 and 34BH11 have been less successful in three successive assessments for CK-LMW. The most robust markers for CK-LMW are the clones C51 and DC10, followed by the clone 5D3. The literature and vendors’ information on clone C51 give conflicting information about the reactivity with CK types 8 and CK18. In an analysis in the NordiQC laboratory, the reaction pattern of clone C51 is identical with CK18 antibodies like clone DC10 and different from Abs reacting with CK8.
This was the fifth assessment of CK-LMW in NordiQC. Identical pass rates has been achieved in the last two runs as shown in table 3.

Table 3: Pass rates in five NordiQC tests for CK-LMW

In the previous assessments of CK-LMW (run 16 and 20), a total of 61 laboratories obtaining an insufficient result have been given specific recommendations how to improve their protocol. 47 laboratories submitted a new stain in the subsequent run. 26 followed the recommendation, of which 20 improved to good or optimal (77 %). 19 laboratories did not follow the recommendation, and only 3 of these (16 %) obtained a sufficient staining in the subsequent run.

Conclusion
The mAb clones DC10, 5D3 and C51 seem to be robust and sensitive Abs for CK-LMW and should replace the old Abs clones CAM 5.2 and 35βH11. HIER, preferably in an alkaline buffer, seems mandatory for optimal performance for all three recommended clones.
Liver is an appropriate control for CK-LMW: The majority of hepatocytes shall show an at least moderate staining with an enhancement along the cell membranes.