slide to be stained for
Ewing sarcoma, 2. Peripheral primitive
neuroepithelial tumour (pPNET), 3. Granulosa cell tumour, 4.
Synovial sarcoma, 5. Tonsil, 6. Liver.
Criteria for assessing a
staining as optimal included: A moderate
to strong distinct predominantly membranous staining of basal and
parabasal squamous epithelial cells and lymphocytes in the tonsil,
and a moderate to strong distinct
predominantly membranous staining the Ewing sarcoma, pPNET, synovial sarcoma and granulosa cell
tumour. No reaction should be seen in the liver.
41 laboratories submitted stains. At the assessment 5 achieved
optimal staining (12 %), 13 good (32 %), 12 borderline (29 %) and 11
(27 %) poor staining.
The following Abs were used:
mAb clone 12E7 (DakoCytomation, n=35)
mAb clone H036-1.1 (Cell Marque, n=2; Ventana, n=2; Novocastra, n=1)
mAb clone 0-13 (Signet, n=1)
In this assessment optimal staining could only be obtained with the
mAb clone 12E7. However, only 5 out of 35
(14 %) using this clone achieved an optimal staining.
In the optimal protocols with clone 12E7, all used HIER with
Tris-EDTA/EGTA pH 9 as the heating buffer. The mAb clone 12E7 was
typically used in the range of 1:25 – 1:100.
All stains assessed as suboptimal were either generally too weak or
false negative in one or more of the specimens in the multi-block.
Almost all laboratories were able to detect CD99 in the Ewing
sarcoma, whereas the synovial sarcoma, the pPNET and especially the
granulosa cell tumour were only weakly labelled or completely
negative with suboptimal protocols.
In the optimal staining only, basal and parabasal squamous
epithelial cells of the tonsil were identified displaying a distinct
membranous staining, indicating that these cells might serve as an
appropriate positive control for the detection of CD99.
The most frequent causes of insufficient stainings were (often in
- Inappropriate choice of HIER buffer (espc.
citrate pH 6)
- Omission of epitope retrieval
- Too low concentration of the primary Ab
- Inappropriate choice of primary Ab.