 The
slide to be stained for ER comprised:
1. Uterine cervix, 2. Lobular Breast carcinoma
with 80 - 100% positivity, 3. Breast fibrocystic
disease, 4-6. Ductal Breast carcinoma with the following ER status 4:
negative, 5: 40 – 60 % and 6: 80 – 100 % positivity as defined in
four reference laboratories.
(the
block is identical to the multitissue block used in run B1 2006).
All tissues were fixed in 10% neutral buffered formalin.
Criteria for assessing an ER staining as optimal included:
- A strong, distinct nuclear staining of
both the columnar and squamous epithelial cells and most stromal
cells (with the exception of endothelial cells
and lymphoid cells) in the uterine cervix.
- A strong, distinct nuclear staining of
the epithelial cells in the fibrocystic disease.
- A strong, distinct nuclear staining of
the appropriate proportion of neoplastic cells in the ductal breast carcinomas no. 5 and 6
and in the breast lobular carcinoma.
- No nuclear staining in the ductal breast carcinoma no. 4 but a week, focal positivity of
the stromal cells.
- No more than a weak cytoplasmic reaction in cells with
strong nuclear staining.
73 laboratories participated in the
assessment. 38 achieved optimal marks (52 %), 23
good (32 %), 11 borderline (15 %) and 1 (1 %) poor marks.
The following antibody clones were used:
rmAb clone SP1 (NeoMarkers, n=13; Ventana, n=11; Diagnostic
Biosystems; n=1).
mAb clone 1D5 (Dako n=24; Immunotech, n=1).
mAb clone 6F11 (Novocastra, n=12; Ventana, n=8; Monosan,
n=1).
mAb clones 1D5 + ER-2-123 (Dako ER/PR pharmDx, n=2).
Optimal staining for ER in this assessment was
obtained with the rmAb clone SP1 (16 out of 25, 64%), the
mAb clone 1D5 (7 out of 25, 28%) and the mAb clone 6F11
(15 out of 21, 71%). All 38 optimal protocols were based on Heat
Induced Epitope Retrieval (HIER).
SP1: all protocols
resulting in an optimal staining were based on HIER using
either Tris-EDTA/EGTA pH 9 (8 out of 10 laboratories using
this obtained an optimal mark), Cell Conditioning1 (Benchmark,
Ventana, 6 out of 11 laboratories using this obtained an optimal
mark), EDTA/EGTA pH 8 (only one laboratory used this buffer
and got the an optimal mark) or Citrate buffer pH 6 (1 out of 2
laboratories using this obtained an optimal mark). The Ab
was typically used in the range of 1:25 - 1:100 or applied as a
Ready-To-Use (RTU) Ab. Using these settings 23 out of 25 (92 %) obtained a
sufficient staining.
1D5: all protocols
resulting in an optimal staining were based on HIER using
Tris-EDTA/EGTA pH 9 (7 out of 14 using this obtained optimal
marks). The Ab was typically diluted in the range
of 1:50 – 1:100. Using these settings 16 out of 20 (80 %) obtained a
sufficient staining.
6F11: all protocols
resulting in an optimal staining were based on HIER using
either Tris-EDTA/EGTA pH 9 (8 out of 9 laboratories using
this obtained an optimal mark), Cell Conditioning1
(Benchmark, Ventana, 5 out of 9 laboratories using this obtained an
optimal mark), Bond Epitope Retrieval Solution 2 (Bond,
Vision Biosystems, only one laboratory used this buffer and got an optimal
mark) or Citrate buffer pH 6 (only one laboratory
used this buffer and got an optimal mark). The Ab was
typically used in the range of 1:50 – 1:300 depending on the total
sensitivity of the protocol employed, or applied as a RTU
Ab. Using these settings 18 out of 20 (90 %) obtained a sufficient
staining.
The most frequent causes of insufficient
staining were:
- Insufficient HIER (citrate pH 6 for the clone 1D5 and/or too short
heating time)
- Too low concentration of the primary antibody
- Use of a biotin based detection system.
The tissues circulated in this run were the same
as used
in run B1 2006
allowing a direct
comparison. In both runs false negatives
as well as false positives are seen. In the current run the prevalent
feature was a
false negative reaction especially the lobular carcinoma, typically seen in protocols
using HIER in Citrate pH 6 or too short HIER time. The false
positive reaction was typically seen in the lobular breast carcinoma
(compromising the interpretation of the specific nuclear reaction of
ER) in protocols based on a biotin based
detection system in combination with HIER espc. in an alkaline
buffer. However compared to run B1, the number of laboratories
obtaining false positive reactions was reduced from 9 to 4, in
parallel with the declining number of laboratories using
biotin based detection systems.
This was the 5th NordiQC assessment of ER. The proportion of
sufficient results has increased relatively constant through the
individual assessments. Many factors contribute to the improvement
but especially the focus on 1. HIER, 2. Ab. concentration and 3. Use
of non-biotin based detection systems seems to be the main
parameters for an optimal demonstration of ER. If these parameters
are individually adjusted for the Ab, the choice of clone is of less
importance. However the rmAb SP1 seems to be slightly more robust
than both 1D5 and 6F11. Using the individual protocol settings as
listed to obtain an optimal staining for each of the 3 clones, 28
out of 30 laboratories (93%) using SP1 obtained a sufficient result
for ER in run B1 and B3, compared to 34 out of 41 laboratories using
6F11 (83%) and 32 out of 39 laboratories (82%) using 1D5.
The uterine
cervix seems to be an appropriate control for the
evaluation of the sensitivity of the ER staining. In an optimal
protocol almost all squamous epithelial cells shall show a distinct nuclear reaction and also the majority of
stromal cells shall be demonstrated.
Conclusion
The mAb clones 6F11, 1D5 and the rmAb SP1 are all well performing
Abs for ER. HIER is mandatory. For optimal demonstration an
alkaline buffer is preferable. The concentration of the Ab must be
carefully calibrated on an appropriate control such as the uterine
cervix. |
