The slide to be stained for ER comprised the following five tissues:

*ER-status and staining pattern as characterized by NordiQC reference laboratories using the mAb clone 6F11 and the rmAb clone SP1.

All tissues were fixed in 10% neutral buffered formalin for 24 – 48 hours and processed according to the “Consensus recommendations on estrogen receptor testing in breast cancer by immunohistochemistry”, AIMM, vol 16, no. 6, 2008.

Criteria for assessing an ER staining as optimal included:

• A moderate to strong, distinct nuclear staining of both the columnar and squamous epithelial cells and most of the stromal cells (with the exception of endothelial cells and lymphoid cells) in the uterine cervix.

• An at least weak to moderate distinct nuclear staining of the appropriate proportion of the neoplastic cells in the breast ductal carcinoma no. 4.

• A strong distinct nuclear staining of the appropriate proportion of the neoplastic cells in the breast ductal carcinoma no. 5.

• No nuclear staining in the neoplastic cells in the breast carcinoma no. 2 and 3 and no more than a weak cytoplasmic reaction in cells with a strong nuclear staining.

A cytoplasmic reaction in the breast ductal carcinoma no. 2 was accepted when using the mAb clone 1D5, as this did not influence the interpretation.

144 laboratories participated in this assessment. 74 % achieved a sufficient mark. In table 1 the antibodies (Abs) used and marks are summarized.

Table 1. Abs and assessment marks for ER, run B8

1) Proportion of sufficient stains (optimal or good), 2) Proportion of sufficient stains with optimal protocol settings only, see below.

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

Concentrated Abs
rmAb SP1: The protocols giving an optimal result were all based on heat induced epitope retrieval (HIER) using Tris-EDTA/EGTA pH 9 (2/12)*, Target Retrieval Solution (TRS) pH 9 (EnVision FLEX TRS high pH, Dako, (4/10), Cell Conditioning 1 (BenchMark, Ventana) (3/4), Bond Epitope Retrieval Solution 2 (Bond, Leica) (1/2), EDTA/EGTA pH 8 (1/1)* or Citrate pH 6 (3/7) as retrieval buffer. The rmAb was typically diluted in the range of 1:25– 1:250 depending on the total sensitivity of the protocol employed. Using these protocol settings 25 out of 35 (71 %) laboratories produced a sufficient staining (optimal or good).
* (number of optimal results/number of laboratories using this buffer)

mAb 6F11: the protocols giving an optimal result were all based on HIER using Tris-EDTA/EGTA pH 9 (4/8), TRS pH 9 (EnVision FLEX TRS high pH, Dako, (4/6), Bond Epitope Retrieval Solution 2 (Bond, Leica) (3/6), Cell Conditioning 1 (BenchMark, Ventana) (1/4) or Citrate pH 6 (2/3) as retrieval buffer. The mAb was typically diluted in the range of 1:30– 1:400 depending on the total sensitivity of the protocol employed. Using these protocol settings 21 out of 25 (84 %) laboratories produced a sufficient staining (optimal or good).

mAb 1D5: the protocols giving an optimal result were all based on HIER using Tris-EDTA/EGTA pH 9 (1/12) or TRS pH 9 (EnVision FLEX TRS high pH, Dako, (3/8) as retrieval buffer. The mAb was typically diluted in the range of 1:50– 1:200 depending on the total sensitivity of the protocol employed. Using these protocol settings 13 out of 17 (76 %) laboratories produced a sufficient staining (optimal or good).

Ready-To-Use Abs
rmAb clone SP1, prod. no 790-4324/25, Ventana: The protocols giving an optimal result were all based on HIER on the BenchMark XT or the Ultra using Cell Conditioning 1, mild or standard, an incubation time of 16-32 min in the primary Ab and iView or ultraView as the detection system. 1 laboratory used the Ab with HIER in Citrate pH 6, UltraView + amplification using the Nexes. Using these protocol settings 38 out of 39 (97 %) laboratories produced a sufficient staining.

rmAb clone SP1, prod. no IR151, Dako: The protocol giving an optimal result was based on HIER using TRS pH 9 (EnVision FLEX TRS high pH) for 20 min in the PT-Link, an incubation time of 20 min in the primary Ab and EnVision Flex (K8000) as the detection system. Using these protocol settings 4 out of 6 (67 %) laboratories produced a sufficient staining.

mAb clone 1D5, prod. no IS654, Dako: The protocol giving an optimal result was based on HIER using TRS pH 9 (EnVision FLEX TRS high pH) for 20 min in the PT-Link, an incubation time of 20 min in the primary Ab and EnVision Flex (K8000) as the detection system.

The most frequent causes of insufficient stainings were:
- Too low concentration of the primary antibody.
- Insufficient HIER (use of citrate pH 6.0 and/or too short efficient heating time)
- Excessive retrieval impairing the morphology

In this assessment the prevalent feature of an insufficient staining was a general too weak reaction or complete false negative reaction especially in the ductal carcinoma no. 4 with 60-80% positivity. This pattern was seen in 89 % (33 out of 37) of the insufficient results. As found in the previous runs the uterine cervix could be used as an appropriate control and critical stain quality indicator for the ER staining. In the optimal protocols almost all the epithelial cells throughout the layers of the squamous epithelium and in the glands showed a moderate to strong and distinct nuclear reaction compared to the protocols giving insufficient results in which both the proportion of positive cells and the intensity was significantly reduced. In this run, all the 3 most widely used Abs for ER, the mAb clones 1D5 and 6F11 and the rmAb clone SP1 could be used to obtain an optimal staining. An optimal staining could both be obtained, when the Abs were used as a concentrate and applied in an in-house protocol or applied as a Ready-To-Use format.
In table 2 the overall performance and accumulated pass rates of the three most widely used markers for ER in the NordiQC assessments are listed.
As observed in the previous assessment of ER an insufficient staining could also be due to excessive HIER typically as a consequence of too long heating time and/or too high temperature hampering the morphology and thus complicating the interpretation. This pattern was seen in 11 % of the insufficient results.

Table 2. Results for the three most used Abs in eight ER tests in  NordiQC

*Runs 8, 10, 13, B1, B3, B5, B7, B8. ** HIER settings and dilution range of the Ab in all assessments giving an optimal result.
   
This was the 8' assessment of ER in the NordiQC breast module and a relative constant proportion of sufficient results have been obtained in the previous 6 runs as shown in table 3, despite many new participants:

Table 3: Sufficient over-all results for ER in the eight NordiQC runs

Conclusion
The mAb clone 6F11 and the rmAb SP1 seem to be the most robust Abs for ER. HIER is mandatory, preferable in an alkaline buffer and shall be performed to provide an optimal balance between sensitivity and preserved morphology. The concentration of the Ab must be carefully calibrated on an appropriate control such as the uterine cervix in which both the epithelial cells and most stromal cells shall show a strong distinct nuclear reaction with minimal cytoplasmic reaction.