slide to be stained for
4-5: Gastrointestinal stromal tumour (GIST).
Criteria for assessing a
staining as optimal included:
- A strong and distinct predominantly
membranous but also cytoplasmic staining of mast cells in all
specimens and the appendiceal cajal cells as well as the two GISTs
- Generally a negative staining of all
other cells and in particular smooth muscle cells. However, some
spindle shaped cells in one of the samples of desmoid revealed a
staining in some laboratories while not in others, in a pattern
that could not be associated to the Abs or protocols. As we have
found no explanation for this phenomenon, it has been ignored in the
87 laboratories submitted stained slides. At
the assessment 17 achieved optimal staining (20 %), 56 good (64 %),
10 borderline (12 %) and 4 (5 %) poor staining.
The following Abs were used:
mAb clone 2E4 (NeoMarkers n=1)
mAb clone 9.7 (Ventana n=1)
pAb A4502 (DakoCytomation n=82)
pAb CMA 767 (Cell Marque n=1)
pAb RB-1518-P (NeoMarkers n=1)
pAb SC-168 (Santa Cruz n=1)
Optimal staining in this assessment could only
be achieved with the pAb A4502 (17 out of 82 were optimal).
All protocols resulting in an optimal staining
were based on HIER with the following buffers: Tris-EDTA/EGTA pH 9
(14 out of 54 were optimal), Citrate pH 6 (1 out of 8 was optimal),
TRS pH 9,9 DakoCytomation (1 out of 2 was optimal) and CC1 Ventana
(1 out of 9 was optimal). In the optimal protocols the pAb A4502 was
used in the range of 1:200 – 1:500 depending on the total
sensitivity of the protocol employed.
The combination of the pAb A4502, diluted in the
range of 1:200 – 1:500 and HIER in Tris-EDTA/EGTA pH 9 resulted in
an optimal staining in 14 out of 33 laboratories (42 %).
The most frequent causes of insufficient
staining were (often in combination):
- Too low concentration of the primary antibody
- Too high concentration of the primary antibody
- Omission of HIER
- Apparently inappropriate choice of primary Ab
The prevalent features of insufficient staining
was either a false negative or a false positive reaction in the
specimens. The false negative reaction was characterized by either a
too weak or totally negative reaction of the appendiceal Cajal cells
and the two GISTs. This was typically seen in protocols omitting
HIER. The false positive reaction was typically seen in endothelial
cells, germinal centre cells and smooth muscle cells, typically
appearing with protocols using a too high concentration of the
primary Ab. The false positive reaction was present both in
protocols with HIER and without HIER.
CD117 was also assessed in
run 7, in which 56
laboratories participated. Out of these 38 % (21 laboratories) had
an insufficient staining. Each laboratory was given specific
recommendations to improve their protocol. 19 laboratories, which
obtained an insufficient result in run 7 submitted a new CD117 stain
in run 14. 16 out of these followed the recommendation and 15 of
them (94 %) improved from insufficient to either good or optimal. 3
laboratories did not follow the recommendations and only one
The overall proportion of insufficient staining
was in this run reduced from 38 % in run 7. to 17 % in run 14.
Focusing only on the laboratories participating in both runs (n=51)
the proportion of insufficient staining was reduced from 37 % (n=19)
to 18 % (n=9).