 The
slide to be stained for mammaglobin
comprised:
1. Breast, 2. Skin, 3-5. Breast ductal carcinoma.
All tissues were fixed in 10 % neutral buffered formalin.
Criteria for assessing a mammaglobin staining as optimal included:
- A strong, distinct cytoplasmic reaction
in scattered ductal epithelial cells and in the apocrine
metaplastic cells of the breast.
- A moderate to strong, distinct
cytoplasmic reaction of the majority of the epithelial cells of
the eccrine sweat glands in the skin.
- A strong, distinct cytoplasmic reaction
of virtually all the neoplastic cells of the breast carcinoma
no. 3 and the majority of neoplastic cells of the breast
carcinoma no. 4.
- At least a weak to moderate cytoplasmic
and focally a dot-like reaction in the majority of the
neoplastic cells the breast carcinoma no. 5.
- No more than a weak background reaction
in the vicinity of the positive cells (antigen diffusion).
23 laboratories participated in the
assessment. 83 % achieved a sufficient mark. The results are summarized in Table 1.
Table 1.
Abs and scores for mammaglobin, run 25
|
Concentrated Abs |
N |
Vendor |
Optimal |
Good |
Borderl. |
Poor |
Suff.1 |
Suff. OPS2 |
|
mAb clone
304-1A5 |
14
4 |
Dako
BioLogo |
7 |
8 |
2 |
1 |
83 % |
100 % |
|
pAb
53625 |
1 |
AnaSpec.
Inc. |
0 |
0 |
0 |
1 |
- |
- |
|
Ready-To-Use Abs |
|
|
|
|
|
|
|
|
|
mAb clone 304-1A5 |
2 |
Dako, IR074 |
2 |
0 |
0 |
0 |
- |
- |
|
rmAb clone 31A5 |
2 |
Ventana, 760-4623 |
2 |
0 |
0 |
0 |
- |
- |
|
Total |
23 |
|
11 |
8 |
2 |
2 |
- |
- |
|
Proportion |
|
|
48 % |
35 % |
9 % |
9 % |
83 % |
100 % |
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 304-1A5: The protocols giving an optimal result
were all based on heat induced epitope retrieval (HIER) using
Tris-EDTA/EGTA pH 9 (5/9)*, Target Retrieval Solution pH 9 (EnVision FLEX TRS
high pH, Dako, (1/1) or EDTA/EGTA pH 8 (1/1) as retrieval buffer.
The mAb was typically diluted in the range of 1:50 – 1:400 depending
on the total sensitivity of the protocol employed. Using these
protocol settings 11 out of 11 (100 %) laboratories produced a
sufficient staining (optimal or good).
* (number of optimal results/number of
laboratories using this buffer)
Ready-To-Use Abs
mAb clone 304-1A5, IR074, Dako: The protocols giving an
optimal result were all based on HIER using Target Retrieval
Solution pH 9 (EnVision FLEX TRS high pH) and an incubation time for 20 min
in the primary Ab and EnVision Flex as the detection system. Using
these protocol settings 2 out of 2 (100 %) laboratories produced an
optimal staining.
rmAb clone 31A5, 760-4263, Ventana: The protocols giving an
optimal result were based on HIER using Cell Conditioning 1, mild or
standard and the incubation time for the primary Ab was 32 min. UltraView was used as detection system. Using these protocol
settings 2 out of 2 (100 %) laboratories produced an optimal
staining.
The frequent causes of insufficient staining were:
- Too low or too high concentration of the primary Ab
- Less successful primary 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 breast
carcinoma no. 5, while the two other breast carcinomas were
demonstrated by virtually all participants. Also the number of
positive cells in the normal breast was significantly reduced
compared to the reaction in a sufficient staining. The false
positive staining was characterized as both a nuclear staining in
the breast carcinomas and as a cytoplasmic staining in many cell
types as smooth muscle cells, fibroblasts etc.
Skin was found to be an appropriate control in which the epithelial
cells of the eccrine sweat glands should show an as strong as
possible cytoplasmic reaction, while other cells as e.g. smooth
muscle cells, squamous epithelial cells should be negative. Normal
breast tissue was less useful as control, as the number and
intensity of the ductal epithelial cells varied throughout the
tissue.
Conclusion
The mAb clone 304-1A5 and the rmAb clone 31A5 are both
useful markers for mammaglobin.
HIER - preferable in an alkaline buffer - seems mandatory to obtain
an optimal staining. Skin is recommended as positive control: The
eccrine sweat glands shall show an as strong as positive cytoplasmic
reaction, while all other cells shall be negative. |
Fig. 1a.
Optimal staining for mammaglobin using the mAb clone 304-1A5
optimally calibrated and with HIER.
Left: Skin: The majority of the epithelial cells of the eccrine
sweat glands show a distinct cytoplasmic reaction.
Right: Breast: The apocrine metaplastic cells and few ductal
epithelial cells show a distinct cytoplasmic reaction.
Also compare with Figs. 2a & 3a – same protocol. |
Fig. 1b.
Insufficient staining for mammaglobin using the mAb clone 304-1A5
too diluted.
Left: Skin: Only scattered epithelial cells of the eccrine sweat
glands show a weak cytoplasmic reaction.
Right: Breast: The epithelial cells are virtually negative and only extracellular mucus is demonstrated. Also compare with Figs. 2b & 3b
– same protocol. |
|
Fig. 2a.
Optimal staining for mammaglobin of the breast ductal carcinoma no. 4
using same protocol as in Fig. 1a. The majority of the neoplastic
cells show a moderate to strong and distinct cytoplasmic reaction.
Also note a weak nuclear reaction is seen in some stromal cells –
this was seen in the majority of the results and is probably due to
absorption of antigen diffusion from the tumour cells. |
Fig. 2b.
Insufficient staining mammaglobin of the breast ductal carcinoma no.
4 using same protocol as in Fig. 1b. The neoplastic cells are
demonstrated, but both the intensity and proportion is reduced -
same field as in Fig. 2a. Also compare with Fig. 3b – same protocol. |
