slide to be stained for
3. Spleen with acute myeloid leukaemia, 4.
Lymph node with Hodgkin’s lymphoma mixed cellularity (MC) type, 5.
Lymph node with Hodgkin’s lymphoma nodular sclerosing (NS) type.
Criteria for assessing a
staining as optimal included:
- A strong and distinct predominantly
membranous staining as well as dot-like (Golgi) staining of
Hodgkin and Reed-Sternberg cells in both cases of Hodgkin’s
- A strong membranous and cytoplasmic
staining of the acute myeloid leukaemia
- A strong predominantly membranous
reaction of the breast duct epithelium and renal proximal tubules
84 laboratories submitted stained slides. At
the assessment 25 achieved optimal staining (30 %), 26 good (31 %),
15 borderline (18 %) and 18 (21 %) poor staining.
The following Abs were used:
mAb clone C3D-1 (DakoCytomation, n=46)
mAb clone MMA (Becton Dickinson, n=23; Ventana, n=4; NeoMarkers, n=3)
mAb clone BY87 (Novocastra, n=4; Ventana, n=2)
mAb clone H198 (Becton Dickinson, n=1)
mAb clone Tu9 (Quartett Immunodiagnostika, n=1)
Optimal stains in this assessment were
obtained only with the clones MMA (16 out of 29 were
optimal), and C3D-1 (9 out of 46 were optimal).
With clone MMA all optimal protocols
was based on HIER using either Tris-EDTA/EGTA pH 9 (15 out of 21
were optimal) or Citrate pH 6 (1 out of 4 was optimal).
With clone 3D-1 all optimal protocols were based on HIER
using Tris-EDTA/EGTA pH 9 (9 out of 25 were optimal).
MMA was typically used in the range of 1:10 – 1:50 and C3D-1
in the range of 1:5 – 1:25 depending on the total sensitivity of the
The combination of clone MMA, diluted in the range of 1:10 –
1:50 and HIER in Tris-EDTA/EGTA pH 9 resulted in an optimal staining
in 15 out of 19 laboratories (79 %), while the combination of clone
C3D-1, diluted 1:5 – 1:25 and HIER in Tris-EDTA/EGTA pH 9 resulted
in an optimal staining in 9 out of 19 laboratories (47 %).
The most frequent causes of insufficient
staining were (often in combination):
- Too low concentration of the primary antibody
- Insufficient HIER - too short heating time
- Inappropriate choice of HIER buffer – citrate pH 6 combined with a
low total sensitivity of the protocol
- Apparently inappropriate choice of primary Ab
The majority of laboratories were able to
detect CD15 in AML and in the neutrophil granulocytes, whereas the
demonstration of CD15 in the two Hodgkin’s lymphomas was much more
difficult and only achieved in the correctly calibrated protocol. In
the optimal stain the epithelial cells of the proximal tubules
typically showed a strong and distinct predominantly membranous
reaction, indicating that these cells may serve as a reliable
control – using a biotin based detection system care should be taken
in the interpretation as cytoplasmic endogenous biotin mimics the
true staining reaction.
CD15 was also assessed in run 10. In that
run 71 laboratories participated out of which 50 % (35 laboratories)
had an insufficient staining. Each laboratory was given a specific
recommendation to improve their protocol. 33 laboratories, which
obtained an insufficient result in run 10 submitted a new CD15 stain
in run 14. 17 out of the 33 laboratories followed the recommendations,
and 12 of these (71 %) improved the assessment from insufficient to
either good or optimal. 16 laboratories did not follow the
recommendation and only 2 (12 %) improved.
The overall proportion of insufficient
staining was in this run reduced from 50 % in run 10 to 40 %, but as
the proportion of insufficient staining still is relative high, CD15
will be repeated in 2006.