 The
slide to be stained for
CD117 comprised:
1. Appendix, 2. Desmoid tumour, 3-5. Gastrointestinal stromal tumour
(GIST).
All tissues were fixed in 10% neutral buffered formalin.
Criteria for assessing a CD117 staining as
optimal included:
- A strong and distinct, predominantly
membranous but also cytoplasmic staining of the Cajal cells in
the appendiceal muscularis propria.
- A moderate to strong, distinct staining
of virtually all the neoplastic cells of the three GISTs.
- A negative reaction of the desmoid
tumour.
- A strong predominantly membranous
staining of the mast cells in all specimens.
- A negative staining of all
other cells (in particular smooth muscle cells). However, a weak
reaction in the endothelial cells and peripheral
nerves was accepted.
128 laboratories participated in this
assessment. 81 % achieved a sufficient mark. In table 1 the
antibodies (Abs) used and marks are summarized.
Table 1.
Abs and scores for CD117, run 26
|
Concentrated Abs |
N |
Vendor |
Optimal |
Good |
Borderl. |
Poor |
Suff.1 |
Suff. OPS2 |
|
pAb A4502 |
113 |
Dako |
29 |
70 |
12 |
2 |
88% |
92% |
|
pAb RB-9038-P |
2 |
NeoMarkers |
0 |
0 |
2 |
0 |
- |
- |
|
rmAb clone
YR145 |
2
1
1 |
Epitomics
Cell Marque
Master
Diagnostica |
3 |
0 |
1 |
0 |
- |
- |
|
Ready-To-Use Abs |
|
|
|
|
|
|
|
|
|
rmAb clone 9.7 |
8 |
Ventana |
0 |
1 |
7 |
0 |
12% |
- |
|
mAb clone T595 |
1 |
Menarini |
0 |
0 |
0 |
1 |
- |
- |
|
Total |
128 |
|
32 |
71 |
22 |
3 |
- |
- |
|
Proportion |
|
|
25% |
56% |
17% |
2% |
81% |
92 % |
1) Proportion of sufficient stains
(optimal or good), 2) Proportion of sufficient stains with optimal
protocol settings only, see below.
The following central protocol parameters were used to obtain an
optimal staining:
Concentrated Abs
pAb A4502: The protocols giving an optimal result were
all based on heat induced epitope retrieval (HIER) using an alkaline
buffer as Tris-EDTA/EGTA pH 9 (11/32)*, Cell Conditioning 1
(BenchMark, Ventana) (9/24), Target Retrieval Solution pH 9
(EnVision FLEX TRS high pH, Dako, (6/22) or Bond Epitope Retrieval
Solution 2 (Bond, Leica) (3/7) as retrieval buffer. The mAb was
typically diluted in the range of 1:100 – 1:800 depending on the
total sensitivity of the protocol employed. Using these protocol
settings 82 out of 89 (92 %) laboratories produced a sufficient
staining (optimal or good).
* (number of optimal results/number of
laboratories using this buffer)
rmAb clone YR145: all three protocols giving an optimal
result were based on HIER using
either Tris-EDTA/EGTA pH 9 (1/1), EDTA/EGTA pH 8 (1/1) or Cell
Conditioning 1 (BenchMark, Ventana) (1/1). The rmAb was diluted in
the range of 1:200 – 1:800 depending on the total sensitivity of the
protocol employed. Using these protocol settings 3 out of 3 (100 %)
laboratories produced a sufficient staining.
The most frequent causes of insufficient staining were:
- Less successful primary antibody
- Too low or too high concentration of the pAb A4502
- Omission of HIER
- Insufficient HIER (e.g., citrate pH 6.0 or too short heating
time).
In this assessment and in concordance with the results in the
previous NordiQC assessments of CD117, the prevalent features of an
insufficient staining were either a weak/false negative reaction or
a false positive reaction in the tested specimens. A weak/false
negative reaction was observed in 21 out of 25 (84%) of the
insufficient results. These were characterized by a too weak
reaction of the appendiceal Cajal cells (which in the previous
NordiQC assessments have been identified as the most appropriate
positive control for CD117. The false positive reactions (due to a
too high Ab concentration) was
typically seen in the smooth muscle cells of lamina muscularis
propria of the appendix and in the smooth muscle cells of the large vessels. In 3 laboratories the desmoid
tumour was distinctively stained, while the other structures stained as expected
inclusive a negative reaction of the
smooth muscle cells. This aberrant pattern was also noticed in the
previous CD117 run 21, 2007 and is most likely related to a
lot-to-variation of the pAb A4502 (Dako). Both in the previous and
current CD117 run, lot OHO12A has been shown to give
this aberrant reaction in the desmoids tumours.
Using pAb A4502 the peripheral nerves in the appendix
frequently showed a moderate cytoplasmic reaction, most pronounced
when a high concentration (1:20 – 1:300) of the Ab was applied. The reaction pattern was seen
with different HIER settings like HIER in Citrate pH 6.0 and Tris-EDTA pH 9.0 and observed
with various lots of A4502. The nerves were negative in all
protocols based on the rmAb YR145.
This was the fourth assessment of CD117 in NordiQC. A relatively
constant proportion of sufficient results have been seen in the
latest three runs, as shown in table 2:
Table 2:
Sufficient results with CD117 in four NordiQC runs
In the previous assessments of CD117 (run 7, 14 and 21), a total of
61 laboratories obtaining an insufficient result were given
specific recommendations how to improve their protocol for CD117. 47 laboratories submitted a new stain in
a subsequent run. 35 followed the recommendation, of which 28 improved
to good or optimal (80 %). 12 laboratories did not follow the
recommendation, and only 3 of these (25 %) obtained a sufficient staining
in the subsequent run. 4 laboratories changed their entire system
and obtained a sufficient result.
Conclusion
pAb A4502 and rmAb clone YR145 are both recommendable
Abs for CD117 in GIST. As regards A4502, however, some lot-to-lot
variation has been revealed. HIER, preferable in
an alkaline buffer, is mandatory for an optimal result.
Concentration of the primary Ab should be carefully calibrated.
Appendix is an appropriate control for CD117: The Cajal cells must
show a distinct staining reaction, while the smooth muscle cells in
muscularis propria must be negative. |
