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, distinct, predominantly
membranous reaction of the neoplastic cells in the T-cell
lymphomas no. 5 and 7. The T-cell lymphoma no. 6 was CD4 negative
and only remnants of normal T-cells should be demonstrated.
- A strong, distinct, predominantly
membranous staining of the normal helper/inducer T-cells in all of
- A moderate, distinct staining of
macrophages, in particular the germinal centre macrophages in the
tonsils, Kupffer cells in the liver and the endothelial cells of
the liver sinusoids.
- No staining in other cells.
Especially the B-cells should be negative.
- A nuclear reaction observed with the
clone 1F6 was accepted, as this did not interfere with the
59 laboratories participated in the assessment.
18 achieved optimal staining (31 %), 24 good (41 %), 8 borderline
(14 %) and 9 (15 %) poor staining.
The following Abs were used:
mAb clone 1F6 (BioCare n=1, NeoMarkers n=4, Novocastra n=30,
mAb clone 4B12 (NeoMarkers n= 5, Novocastra n=14)
Optimal staining in this
assessment could be obtained with both clone 1F6 (9 out of
clone 4B12 (9 out of 19).
All optimal protocols with
the two Abs were based on HIER. For obtaining an optimal result with
clone 1F6 the following heating buffers could be used:
Tris-EDTA/EGTA pH 9 (6 out of 24 were optimal), EDTA/EGTA pH 8 (2
out of 5 were optimal) and CC1, Ventana (1
out of 7 were optimal).
1F6 was typically either
diluted in the range of 1:10 – 1:25 (in which 7 out of 27 were
optimal) or used as a Ready-To-use Ab (2 out of 6 were optimal).
The relative low rate of
optimal stains might indicate, that other methodological parameters
could affect the staining result. As regards clone 1F6,
according to vendor
recommendations, blocking of endogenous peroxidase in >1 % H2O2
after HIER can be deteriorative to the antigen detected. This
has been confirmed in several NordiQC reference laboratories
(see Figs. 3a-3b). Thus
it is recommended either to use a lower concentration of H2O2,
performing the peroxidase blocking before HIER or omit endogenous
peroxidase blocking at all. 3 out of 3 laboratories omitting
peroxidase blocking or performing the step before HIER obtained good
For obtaining an optimal
result with clone 4B12 the following heating buffers could be used:
Tris-EDTA/EGTA pH 9 (6 out of 14 were optimal), EDTA/EGTA pH 8 (2
out of 3 were optimal) and TRS 9,9, DakoCytomation (1 out of 1 was
optimal). 4B12 was typically diluted in the range of 1:25 – 1:400
(in which 9 out of 17 were optimal).
The most frequent causes of
insufficient staining were (often in combination):
- Too low concentration of the primary
- Insufficient HIER (i.e. using Citrate pH
6 as HIER buffer or a too short HIER time, i.e., < 15 efficient
heating time in a MWO)
- Possibly inappropriate blocking of
endogenous peroxidase with the clone 1F6 (10 laboratories using the
same protocol as used for an optimal staining, obtained an
insufficient result, however, we have not yet obtained data
regarding this question)
The prevalent feature of an
insufficient staining was a too weak and diffuse staining or almost
completely negative reaction of both the neoplastic T-cells and the
normal helper T-cells. A good quality indicator in this assessment
was the germinal centre macrophages, as these cells typically were
demonstrated in a staining assessed as optimal or good, while the
cells were too weak or negative in staining assessed as borderline
Compared to normal helper/inducer
T-cells the macrophages generally express a weak CD4 staining. The
capability to detect these cells verifies the sensitivity of the