Table 1. Abs and assessment marks for PR, run B12

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

The following central protocol parameters were used to obtain an optimal staining:

Concentrated Abs
mAb clone 16: The protocols giving an optimal result were all based on heat induced epitope retrieval (HIER) using either Tris-EDTA/EGTA pH 9 (1/1)*, Target Retrieval Solution (TRS) pH 9 (3-in-1) (Dako)
(2/2)*, Bond Epitope Retrieval Solution 2 (BERS2; Bond, Leica) (5/6)*, BERS1 (Bond, Leica) (1/1)* or EDTA/EGTA pH 8 (1/1)* as the retrieval buffer. The mAb was typically diluted in the range of 1:200 – 1:1,600 depending on the total sensitivity of the protocol employed. Using these protocol settings 10 out of 11 (91 %) laboratories produced a sufficient staining (optimal or good).
*(number of optimal results/number of laboratories using this buffer)

mAb clone PR 636: The protocols giving an optimal result were all based on HIER using either Tris-EDTA/EGTA pH 9 (4/11), TRS pH 9 (Dako) (2/11), TRS pH 9 (3-in-1,Dako) (6/11) or BERS2 (Bond, Leica) (1/4) as the retrieval buffer. The mAb was typically diluted in the range of 1:100 – 1:500 depending on the total sensitivity of the protocol employed. Using these protocol settings 20 out of 29 (69 %) laboratories produced a sufficient staining.

rmAb clone SP2: The protocol giving an optimal result was based HIER using Tris-EDTA/EGTA pH 9 (1/2) as the retrieval buffer. The rmAb was diluted 1:200.

rmAb clone Y85: The protocol giving an optimal result was based on HIER using EDTA/EGTA pH 8 (1/1) as the retrieval buffer. The rmAb was diluted 1:100.

Ready-To-Use Abs
mAb, clone 16 (prod.no. PA0312, Leica/Novocastra). The protocol giving an optimal result was based on HIER in BERS2 (Bond, Leica) for 25 min, an incubation time of 15 min in the primary Ab and Refine (DS9800) as the detection system. Using similar protocol settings 3 out of 3 produced a sufficient staining.

mAb clone PR 636 (prod. no. IS/IR068, Dako): The protocols giving an optimal result were all based on HIER in PT-Link using TRS pH 9 or TRS pH 9 (3-in-1) (heating time 10-30 min at 95-97°C) , an incubation time of 15-30 min in the primary Ab and EnVision Flex (K8000) or Flex+ (K8002) as the detection system. Using these protocol settings 23 out of 27 (85 %) laboratories produced a sufficient staining.

rmAb clone 1E2 (prod. no. 790-2223/4296, Ventana): The protocols giving an optimal result were typically based on HIER using mild or standard Cell Conditioning 1, an incubation time of 20-48 min in the primary Ab and iView (760-091) or UltraView (760-500) as the detection system. Using these protocol settings 56 out of 66 (85 %) laboratories produced a sufficient staining (optimal or good).

The most frequent causes of insufficient stains were:
- Too low concentration of the primary Ab
- Less successful primary Ab

- Insufficient epitope retrieval (too short efficient HIER time and/or use of a non-alkaline HIER buffer).

In this assessment and in concordance with the previous PR assessments in NordiQC, the insufficient results were mainly due to a too weak or completely false negative staining. This pattern was seen in 89 % of the insufficient staining results (65 out of 72 stains). Virtually all the laboratories could demonstrate PR in the ductal breast carcinoma no. 5 with 80-100% positivity and a strong nuclear staining intensity (as established by the NordiQC reference laboratories) whereas the prevalent feature of the insufficient staining was a too weak (< 10% positivity) or entirely false negative staining of the ductal breast carcinoma no. 3 and 4 (with 30-60% positivity and a weak to moderate nuclear staining intensity expected). The insufficient staining reactions were typically caused by a too low concentration of the primary Ab and/or insufficient HIER, but also when using less successful Abs with an apparently low PR affinity. However it also has to be mentioned that a high proportion of stains assessed as insufficient (18 out of 72 protocols) seemed to be based on optimal protocol settings, including a correct titre of an otherwise successful primary Ab, HIER settings and detection kit. Thus, the combination of several suboptimal protocol settings might have caused a reduced sensitivity, e.g., a low concentration of the primary Ab, HIER in a non-alkaline buffer and a detection system with only a moderate sensitivity such as a two-step polymer based kit. For the laboratories using a non-alkaline buffer for HIER the overall pass rate was 34 % (13 out of 38), compared to a pass rate of 74 % when HIER was performed with an alkaline buffer. The same pattern was seen when the pass rates were related to the detection system: With a moderately sensitive detection system, such as a 2-step polymer/multimer based detection system or an avidin-biotin based system, an overall pass rate of 62 % was seen (105 out of 169) compared to 86 % (47 out of 55) for a 3-step polymer/multimer based system.
All 4 stains based on the mAb clone PR 636 and performed on the BenchMark platform (Ventana) were assessed as insufficient, despite of protocol settings similar to the settings giving a sufficient result on other IHC platforms, e.g. the Dako Autostainer.

In 6 % of the insufficient results (4 out of 72 stains) a combination of a too weak staining and impaired morphology was seen. This pattern was typically seen by the use of excessive HIER in combination with a protocol giving a too low sensitivity.
In the remaining 3 insufficient stains (5 %) a false positive staining in the PR negative ductal carcinoma no. 2 was seen. When a false positive reaction was observed in the PR negative tumour also a false positive nuclear reaction was seen in scattered non-epithelial cells as lymphocytes and endothelial cells. The false positive staining was seen for the rmAb clone SP2 (2 out of 5 protocols) and the mAb clone cocktail hPRa2+hPRa3 (1 out of 1 protocol). A false positive nuclear reaction was also observed and described in the previous assessments run B6 and B9 and most likely caused by a combination of inadequate washing in buffer and too high concentration of the primary Ab.

For both the mAb clones 16 and PR 636 the Ready-To-use systems from Leica/Novocastra and Dako, respectively, the obtained pass rates were higher than the pass rates obtained for the same clones used with an in-house validated assay.

As observed in the previous assessments for PR, the uterine cervix seems to be an appropriate control for the evaluation of the sensitivity of the PR staining: With an optimal protocol almost all the columnar epithelial cells, the basal squamous epithelial cells and most of the stromal cells must show a strong and distinct nuclear staining with only a minimal cytoplasmic reaction. Virtually all laboratories obtaining this staining pattern were assessed as sufficient. However, differences regarding the reaction pattern are seen depending on the Ab selected. When using the mAb clone 1A6, the basal squamous epithelial cells are negative and a cytoplasmic reaction is seen in the intermediate and superficial squamous epithelial cells, while the clone PR 636 gives an intense cytoplasmic reaction in the columnar epithelial cells.

This was the 6th assessment of PR in NordiQC. A decrease in the proportion of sufficient results is seen in the two latest runs for PR as shown in table 2:
 

Table 2: Proportion of sufficient results for PR in the six NordiQC runs performed

In the last two assessments for PR many new laboratories have enrolled in the NordiQC programme. In the current run a slightly higher pass rate was observed for the laboratories participating in all three runs from B6 to B12 than for the laboratories participating for the first time in either run B9 or B12: For the laboratories participating for the first time in run B9 or B12 the pass rate was 64 % (86 out of 134), whereas the pass rate was 73 % (66 out of 90 laboratories) for the laboratories participating in all three runs from B6 to B12.

Conclusion
The mAb clones 16, PR 636 and the rmAb clone 1E2 are all well performing and recommendable Abs for PR. In general the Ready-To-Use systems for these three Abs showed a superior pass rate compared to in-house validated assays for PR. Efficient HIER (preferably in an alkaline buffer) is mandatory for an optimal performance and must be carried out to provide an optimal balance between sensitivity and morphology. Uterine cervix is an appropriate control, in which almost all the columnar epithelial cells, the basal squamous epithelial cells and most of the stromal cells must show a strong and distinct nuclear staining reaction with only a minimal cytoplasmic staining.