The slide to be stained for estrogen receptor
alpha (ER) comprised: 1:
lung
adenocarcinoma, 2:
ovarian
serous carcinoma, 3-5:
breast
ductal carcinoma.
Criteria for assessing an ER staining as optimal
included:
A strong and distinct nuclear staining reaction
for ER in almost all of the neoplastic cells in the ductal breast
carcinoma with the high expression of ER, a weak to moderate
staining reaction in most neoplastic cells in the ductal carcinoma
with the low expression of ER, and a heterogeneous staining of
nuclei in
the
serous ovarian carcinoma. A weak cytoplasmic reaction of cells with
strong
nuclear staining was accepted, as was staining of necrotic tissue in
the lung adenocarcinoma.
71 laboratories submitted stainings. At the
assessment 11 achieved optimal staining
(15 %), 21 good (30 %), 15 borderline (35 %) and
14 (20%) poor staining.
All 71 laboratories used either mAb clone 6F11
(36) or 1D5 (35). Clone 6F11 was obtained from Novocastra (27) and
Ventana (9), 1D5 from DakoCytomation (27), Immunotech (7) and
Immunovision (1).
Optimal stainings could be obtained with both
clones. In the optimal protocols all used HIER, primarily based on
MWO with Tris-EDTA/EGTA pH 9 as the heating buffer with a total
heating time (i.e., from starting the MWO until it is turned of) of
20 – 30 min. If citrate pH 6 was used as the heating buffer, the
total heating time in optimal stainings was 20 – 40 min.
Using clone 6F11 optimal stainings was obtained
with a concentration of 1:10 – 1:75. Using 1D5 , the concentration was
in the range of
1:25 – 1:75, depending
on the incubation time
(25 – 60 min.).
In almost all stainings, the ductal
breast carcinoma with the high ER expression was labelled. In cases
where this tumour showed weaker staining, the other breast carcinoma
and the serous carcinoma typically showed false negative reactions.
The probable causes of insufficient stainings
were multiple, and often several suboptimal steps in the individual
protocols could be identified.
In 22 out of the 29 protocols giving an insufficient result, the
total heating time was ≤ 15 min and/or the
antibody concentration was 1:200 – 1:1000.
The most frequent causes of insufficient
stainings were (often in combination):
-Insufficient
HIER (too short
heating time, particularly in combination with Citrate pH6)
-Too
high or too low
concentration of primary antibody.
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