 The
slide to be stained for Gross cystic disease fluid protein-15 (GCDFP)
comprised:
1. Breast, 2. Skin, 3-5. Breast ductal carcinoma.
All tissues were fixed in 10 % neutral buffered formalin.
Criteria for assessing a GCDFP staining as optimal included:
- A strong, distinct cytoplasmic reaction
in scattered ductal epithelial cells and 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 the neoplastic cells of the breast
carcinoma no. 4.
- At least a weak to moderate cytoplasmic
and a dot-like reaction in scattered neoplastic cells of the
breast carcinoma no. 5.
- No more than moderate background reaction
in the vicinity of the positive cells was accepted (antigen
diffusion).
43 laboratories participated in the
assessment. 61 % achieved a sufficient mark. The results are summarized in Table 1:
Table 1.
Abs and scores for GCDFP, run 25
|
Concentrated Abs |
N |
Vendor |
Optimal |
Good |
Borderl. |
Poor |
Suff.1 |
Suff. OPS2 |
|
mAb clone
23A3
|
19
6
2
1
1
1 |
Novocastra
NeoMarkers
Cell Marque
Abcam
Dako
Vector |
11 |
8 |
7 |
4 |
63 % |
85 % |
|
mAb D6 |
4
3
2
1
1 |
Signet
Zymed
ID Labs Biotech.
BioCare
BioGenex |
4 |
1 |
5 |
1 |
46 % |
100 % |
|
Ready-To-Use Abs |
|
|
|
|
|
|
|
|
|
mAb clone
23A3 |
1 |
Novocastra |
0 |
1 |
0 |
0 |
- |
- |
|
rmAb clone
EP1582Y |
1 |
Ventana, 760-4386 |
0 |
1 |
0 |
0 |
- |
- |
|
Total |
43 |
|
15 |
11 |
12 |
5 |
- |
- |
|
Proportion |
|
|
35 % |
26 % |
28 % |
12 % |
61 % |
88 % |
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 23A3: The protocols giving an optimal result were
all based on heat induced epitope retrieval (HIER) using
Tris-EDTA/EGTA pH 9 (5/9)*, Cell Conditioning 1 (BenchMark, Ventana)
(2/8), Bond Epitope Retrieval Solution 2 (Bond, Leica) (1/2), Target
Retrieval Solution pH 9 (EnVision FLEX TRS high pH, Dako, (2/6) or Citrate pH
6 (1/3) as retrieval buffer. The mAb was typically diluted in the
range of 1:20 – 1:100 depending on the total sensitivity of the
protocol employed. Using these protocol settings 17 out of 20 (85 %)
laboratories produced a sufficient staining (optimal or good).
* (number of optimal results/number of
laboratories using this buffer)
mAb clone D6: The protocols giving an optimal result were
based on HIER using Tris-EDTA/EGTA pH 9 (2/2) or Cell Conditioning 1
(BenchMark, Ventana) (2/3) 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 5 out of 5 (100 %) laboratories produced a sufficient
staining (optimal or good).
The most frequent causes of insufficient staining were:
- Too low concentration of the primary Ab.
- Omission of HIER (5/5 laboratories using the clone D6 without HIER
obtained an insufficient mark)
In this assessment the prevalent feature of an insufficient staining
was a general too weak or false negative staining, occasionally
accompanied by an excessive background reaction. The false negative
reaction was in particular observed in the breast carcinoma no. 5,
while a proper staining of the two other breast carcinomas were
demonstrated by the majority of the participants. With mAb clone D6
omission of HIER also gave an intense background reaction in the
normal breast specimen compromising the interpretation. For both mAb
clones D6 and 23A3, the background reaction was significantly
reduced with HIER, and was only seen in the vicinity of the GCDFP
positive glands.
Skin was found to be an appropriate control: The epithelial cells of
the eccrine sweat glands should show an as strong as positive
cytoplasmic reaction, while all other 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 clones 23A3 and D6 are both useful markers for
GCDFP. 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 GCDFP using the mAb clone 23A6 optimally
calibrated and with HIER.
Left: Skin: The majority of the epithelial cells of the eccrine
sweat glands show a strong and distinct cytoplasmic reaction.
Right: Breast: Scattered ductal epithelial cells show a distinct
cytoplasmic reaction and also a strong reaction is seen in the
luminal extracellular mucus. A weak background reaction is seen.
Also compare with Figs. 2a & 3a – same protocol. |
Fig. 1b.
Insufficient staining for GCDFP using the mAb clone D6 with omission
of HIER.
Left: Skin: The majority of the epithelial cells of the eccrine
sweat glands show a moderate cytoplasmic reaction.
Right: Breast: Scattered epithelial cells show a moderate
cytoplasmic reaction, but also a moderate background reaction is
seen.
Also compare with Figs. 2b & 3b – same protocol. |
|
Fig. 2a.
Optimal staining for GCDFP of the breast ductal carcinoma no. 3
using same protocol as in Fig. 1a. Virtually all the neoplastic
cells show a moderate to strong and distinct cytoplasmic reaction
and only a minimal background reaction is seen. |
Fig. 2b.
Insufficient staining GCDFP of the breast ductal carcinoma no. 3
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. |
