slide to be stained for
Tonsil fixed 4 h,
3: Tonsil fixed 48 h,
fixed 96 h,
5-7: Nodal T-cell lymphomas.
Criteria for assessing a
staining as optimal included:
- A strong and distinct predominantly
membranous as well as cytoplasmic granular reaction of the neoplastic cells in
one of the T-cell
lymphomas (no. 6),
whereas the neoplastic cells in the two other T-cell lymphomas should be
- A strong and distinct membranous as well
as cytoplasmic granular staining of the normal suppressor/cytotoxic
T-cells in all of the tonsils and the liver.
- No staining in other cells.
Especially B-cells in the tonsil should be negative
63 laboratories participated in the assessment.
41 achieved optimal staining (66 %), 17 good (26 %), 4 borderline (6
%) and 1 (2 %) poor staining.
The following Abs were used:
mAb clone C8/144B (DakoCytomation n=41, NeoMarkers n=2)
mAb clone 4B11 (Novocastra n=15)
mAb clone 1A5 (Ventana n=3, Novocastra n=1)
mAb clone DK25 (DakoCytomation n=1)
Optimal staining in this assessment was
obtained with following Abs: clone C8/144B (30 out of 42 were
optimal), clone 4B11 (7 out of 15 were optimal) and clone
1A5 (4 out of 4 were optimal).
All optimal protocols were based on HIER
irrespectively of the Ab employed.
With clone C8/144B all of the following
could be used as the heating buffer: Tris-EDTA/EGTA pH 9 (25 out of
30 were optimal), EDTA/EGTA pH 8 (1 out of 2 was optimal),
CC1,Ventana (2 out of 4 were optimal), TRS pH 9.9, DakoCytomation (1
out of 1 was optimal) and Citrate pH 6 (1 out of 4 were optimal).
C8/144B was typically diluted in the range of 1:50 – 1:200.
With clone 4B11 only Tris-EDTA/EGTA pH 9 could be used (7 out
of 12 were optimal). 4B11 was typically diluted in the range of 1:25
With clone 1A5 all of the following could be used as the
heating buffer: CC1, Ventana (2 out of 2 were optimal), Tris-EDTA pH
9 (1 out of 1 was optimal) and EDTA/EGTA pH 8 (1 out of 1 was
optimal). 1A5 could both be used as a Ready-To-Use product (3 out of
3) or as a concentrated Ab diluted 1:80.
The most frequent causes of insufficient
staining were (often in combination):
- Too low concentration of the primary antibody
- Insufficient HIER
The prevalent feature of an insufficient
staining was a too weak and diffuse staining of the normal
suppressor/cytotoxic T-cells as well as neoplastic T-cells. There
was no significant difference in the staining characteristics of the
normal and the neoplastic T-cells, indicating that tonsil can be
used as reliable control, in which both the isolated and grouped
T-cells should be distinctively demonstrated.