The slide to be stained for AMACR/PIN-cocktail comprised:
1. Prostate intraepithelial neoplasia (PIN), 2. Tonsil, 3. Kidney,
4. Prostate hyperplasia, 5- 6. Prostate carcinoma.
All tissues were fixed in 10% neutral buffered formalin.

Criteria for assessing an AMACR staining as optimal included:

• A moderate to strong granular cytoplasmic staining of the epithelial cells lining the renal proximal tubules.
• A moderate to strong cytoplasmic staining of the majority of the neoplastic cells of the PIN and the two prostate adenocarcinomas.
• A negative or only week focal cytoplasmic staining of the epithelial cells of the hyperplastic prostate glands.
• A negative or only week cytoplasmic staining of the stromal cells.

In case of using a PIN cocktail the criteria also included:

AMACR + p63:

• A moderate to strong, distinct nuclear staining in almost all squamous epithelial cells in the tonsil and in the basal cells of the prostate hyperplastic glands and the PIN lesion.

AMACR + p63 + CK high molecular weight (HMW: CK5, CK5/6, CK14)

• A moderate to strong, distinct nuclear and cytoplasmic staining in almost all squamous epithelial cells in the tonsil and in the basal cells of the prostate hyperplastic glands and PIN lesion.

106 laboratories participated in this assessment. 70 used AMACR as a single marker out of which 90 % achieved a sufficient mark. 36 used a PIN-cocktail out of which 89 % achieved a sufficient mark.

In table 1 the antibodies (Abs) for AMACR and marks are summarized.
 

Table 1. Abs and scores for AMACR, run 26

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

Concentrated Abs
rmAb 13H4: The protocols giving an optimal result were all based on heat induced epitope retrieval (HIER) using Tris-EDTA/EGTA pH 9 (13/18)*, Target Retrieval Solution pH 9 (EnVision FLEX TRS high pH, Dako)(9/11), Cell Conditioning 1 (BenchMark, Ventana) (4/14), EDTA/EGTA pH 8 (1/2), Bond Epitope Retrieval Solution 2 (Bond, Leica) (4/4) or Citrate pH 6 (3/7) as retrieval buffer. The rmAb was typically diluted in the range of 1:25 – 1:200 depending on the total sensitivity of the protocol employed. Using these protocol settings 51 out of 53 (96 %) laboratories produced a sufficient staining (optimal or good).
* (number of optimal results/number of laboratories using this buffer)

pAb p504S, CP200: The protocols giving an optimal result were all based on HIER using Tris-EDTA/EGTA pH 9 (1/2) or Target Retrieval Solution pH 9 (EnVision FLEX TRS high pH, Dako) (1/1). The pAb was typically diluted in the range of 1:25 – 1:200 depending on the total sensitivity of the protocol employed. Using these protocol settings both of 2 laboratories produced a sufficient staining.

Ready-To-Use Abs
rmAb clone 13H4, prod. no IR060, Dako: The protocols giving an optimal result were all based on HIER using Target Retrieval Solution pH 9 (EnVision FLEX TRS high pH), an incubation time of 20 or 30 min in the primary Ab and EnVision Flex/Flex+(K8000/K8002) as the detection system. Using these protocol settings 5 out of 5 (100 %) laboratories produced a sufficient staining.

rmAb clone 13H4, prod. no. RM-9130-R7, NeoMarkers: The protocol giving an optimal result was based on HIER using mild Cell Conditioning 1 (BenchMark, Ventana), an incubation time of 32 min in the primary Ab and iView (760-091) as the detection system. Using these protocol settings 1 out of 2 laboratories produced a sufficient staining.

rmAb clone 13H4, prod. no. 504R-17, Cell Margue: The protocol giving an optimal result was based on HIER using standard Cell Conditioning 1 (BenchMark, Ventana), an incubation time of 32 in the primary Ab and Ultra View (760-500) as the detection system.

In table 2 the antibodies (Abs) for a PIN-cocktail and marks are summarized.

Table 2. Abs and scores for PIN-cocktail, run 26

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:

In-house PIN-cocktails based on concentrated Abs
rmAb clone 13H4 + mAb clone 4A4: The protocols giving an optimal result were all based on heat induced epitope retrieval (HIER) using Tris-EDTA/EGTA pH 9 (2/3)*, Target Retrieval Solution pH 9 (EnVision FLEX TRS high pH, Dako) (2/2), or Cell Conditioning 1 (BenchMark, Ventana) (2/4) as retrieval buffer. The rmAb clone 13H4 was typically diluted in the range of 1:25 – 1:1,000, whereas the mAb clone 4A5 was diluted 1:50 – 1:1.000 depending on the total sensitivity of the protocol employed. Using these protocol settings 10 out of 10 (100 %) laboratories produced a sufficient staining (optimal or good).
* (number of optimal results/number of laboratories using this buffer)

rmAb clone 13H4 + mAb clone 4A4 + mAb clone D5/16 B4: The protocols giving an optimal result were all based on HIER using Tris-EDTA/EGTA pH 9 (1/2)*, Target Retrieval Solution pH 9 (EnVision FLEX TRS high pH, Dako) (1/2), or Cell Conditioning 1 (BenchMark, Ventana) (1/1) as retrieval buffer. The rmAb clone 13H4 was typically diluted in the range of 1:400 – 800, mAb clone 4A4 1:200 – 1:800 and the clone D5/16 B4 1:100 – 1:800 depending on the total sensitivity of the protocol employed. Using these protocol settings 5 out of 5 (100 %) laboratories produced a sufficient staining (optimal or good).

rmAb clone 13H4 + mAb clone (4A4 + Y4A3) + mAb clone D5/16 B4: The protocol giving an optimal result was based on HIER using Tris-EDTA/EGTA pH 9 as retrieval buffer. The rmAb/mAb/mAb was diluted  1:200/1:4.000/1:100.

rmAb clone 13H4 + mAb clone 4A4 + mAb clone XM26: The protocol giving an optimal result was based on HIER using Cell Conditioning 1 (BenchMark, Ventana) as retrieval buffer. The rmAb/mAb/mAb was diluted  1:100/1:100/1:100.

rmAb clone 13H4 + mAb clone 4A4 + mAb clone 34BE12: The protocol giving an optimal result was based on HIER using Cell Conditioning 1 (BenchMark, Ventana) as retrieval buffer. The rmAb/mAb/mAb was diluted 1:50/1:25/1:100.

PIN-cocktails based on Ready-To-Use Abs
mAb clone 4A4 +pAb P504S, prod. no. PPM201, BioCare: The protocols giving an optimal result were all based on HIER using Tris-EDTA/EGTA pH 9 (2/2)* or Target Retrieval Solution pH 9 (EnVision FLEX TRS high pH, Dako) (1/2) an incubation time of 20 or 25 min in the primary Ab and EnVision (K8002/K5007) as the detection system. Using these protocol settings 4 out of 4 laboratories produced a sufficient staining.
* (number of optimal results/number of laboratories using this buffer)

rmAb clone 13H4 + mAb clone 4A4 + mAb clone 34BE12, prod. no. PIN002-G, Biologo: The protocol giving an optimal result was based on HIER using Target Retrieval Solution pH 9 (EnVision FLEX TRS high pH, Dako) an incubation time of 60 min in the primary Ab and EnVision (K5007) as the detection system.

mAb clone 4A4 +mAb clone XM26 + mAb clone LL002 + pAb P504S, prod. no. PPM225DSAA (PIN4), BioCare: The protocol giving an optimal result was based on HIER using Tris-EDTA/EGTA pH 9 an incubation time of 30 min in the primary Ab and MACH2 double stain as the detection system.


The most frequent causes of insufficient stains were:
- Too low or too high concentration of the primary Ab for AMACR
- Too low concentration of the primary Ab for p63 in an in house PIN cocktail

In this assessment and in concordance to the previous assessment of AMACR the prevalent feature of an insufficient staining was a too weak or false negative staining of the epithelial cells in the prostate carcinomas and PIN lesion primarily due to a too low concentration of the primary Ab. In only 2 stains, the insufficient result was caused by a false positive reaction (diffuse background reaction and cytoplasmic staining of the normal prostate epithelial cells). In order to identify a correct calibration of the immunohistochemical protocol for AMACR, kidney appeared to be a suitable control: In the optimally calibrated protocols virtually all the epithelial cells of the proximal tubules showed a strong and distinct granular staining, indicating that these cells may serve as a reliable positive critical stain quality indicator (CSQI) for AMACR. However, kidney is only a reliable positive control for AMACR, when a non-biotin based detection system is applied (as the non-specific reaction of endogenous biotin in the renal tubules mimics the specific staining reaction). As negative control for AMACR, a normal prostate is recommendable: The epithelial cells must be negative or only show a focal staining and the stromal cells must be negative.

In the assessment 35 laboratories used a PIN-cocktail based on 2 – 4 markers. All were based on AMACR and p63, and 11 laboratories also applied a marker for CK HMW. In this context it should be notified that the p63 clones 4A4 and Y4A3 are not CE IVD labelled but sold for Research-Use-Only purpose. There was no significant difference in the performance and staining results regarding the composition of the PIN-cocktail. The PIN-cocktail reactions were visualized both by a single chromogene (such as DAB) or by a dual chromogene system (DAB and Fast Red). In general it is advisable to use different chromogenes to identify and differentiate the individual reactions in a double staining procedure, but as the markers in a PIN-cocktail are located in different compartments (nuclei versus cytoplasm) in the individual epithelial cells of the prostate, and in different cell layers (luminal versus basal) it is possible to use one chromogene to simplify the protocol set-up.

This was the second assessment of AMACR/PIN-cocktail in NordiQC. A constant proportion of sufficient results has been seen as shown in table 2:

Table 2: Sufficient results with AMACR/PIN-cocktail in the two NordiQC runs

Conclusion
The rmAb clone 13H4 and the pAb p504S, CP200 are both recommendable AMACR Abs. They can be used single or in PIN-cocktails together with, e.g., p63 and CK-HMW. HIER is mandatory for an optimal performance. The AMACR staining must be calibrated carefully. Normal prostate is recommendable as negative control and normal kidney as positive control.