 The
slide to be stained for
Prostein
comprised:
1. Appendix, 2. Kidney, 3. Prostate hyperplasia, 4 – 6. Prostate
adenocarcinoma.
(same tissue block as used for PSA)
All tissues were fixed in 10% neutral buffered formalin.
Criteria for assessing a P501S staining as
optimal included:
- A moderate to strong granular cytoplasmic
staining of the epithelial cells of the hyperplastic prostate
glands.
- A moderate to strong granular cytoplasmic
staining of the majority of the neoplastic cells of the three
prostate adenocarcinomas.
- A negative staining in all other cells
such as the epithelial cells in the kidney and appendix.
11 laboratories participated in this
assessment. 73 % achieved a sufficient mark. In table 1 the
antibodies (Abs) used and marks are summarized.
Table 1.
Abs and
assessment marks
for P501S, run 27
|
Concentrated Abs |
N |
Vendor |
Optimal |
Good |
Borderl. |
Poor |
Suff.1 |
Suff. OPS2 |
|
mAb clone 10-E3 |
10 |
Dako |
3 |
5 |
2 |
0 |
70 % |
100 % |
|
Ready-To-Use Abs |
|
|
|
|
|
|
|
|
|
mAb
clone 10-E3,
N1610 |
1 |
Dako |
0 |
0 |
1 |
0 |
- |
- |
|
Total |
11 |
|
3 |
5 |
3 |
0 |
- |
- |
|
Proportion |
|
|
27 % |
46 % |
27 % |
0 % |
73 % |
- |
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
mAb clone 10-E3: the protocols giving an optimal result were
all based on heat induced epitope retrieval (HIER) using
Tris-EDTA/EGTA pH 9 (3/5)* as the retrieval buffer. The mAb was
typically diluted in the range of 1:10– 1:200 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).
* (number of optimal results/number of
laboratories using this buffer)
The causes of insufficient stains were:
- Too low concentration of the primary Ab
- Too high concentration of the primary Ab (incl the Ready-To-Use Ab).
In this assessment the prevalent feature of an
insufficient staining was either a generally too weak staining or a
false positive staining. The former was in particular observed in
the prostate carcinoma no. 4, while the two other prostate
carcinomas were demonstrated by virtually all participants. The
false positive staining was seen as a moderate diffuse cytoplasmic
staining in e.g. the stromal cells in the prostate specimens but
also in the epithelial cells in the kidney and the
appendix. For unexplained reasons plasma cells in 2 protocols based
on HIER in Target Retrieval Solution,
low pH (pH 6.1, Dako) showed a
distinct cytoplasmic reaction. These results were assessed as good.
Prostate was found to be an appropriate
positive control in which the epithelial cells must show an as
strong as possible granular cytoplasmic reaction, while other cells
as e.g. the stromal smooth muscle cells must be negative.
Conclusion
The mAb clone 10-E3 is a recommendable Ab for P501S.
HIER in an alkaline buffer seems mandatory to obtain an optimal
staining. Normal prostate is recommended as positive control: The
glandular epithelial cells must show an intense granular cytoplasmic reaction, while all other cells
should be
negative. |
