|
 The
slide to be stained for
Bcl-2 comprised: 1: Tonsil fixed for 24 h., 2: Tonsil fixed for
168 h., 3 - 5: Follicular lymphoma.
All tissues were fixed in 10% neutral buffered formalin.
Criteria for
assessing a Bcl-2 staining as optimal included: In both tonsils a
moderate to strong distinct cytoplasmic staining of the peripheral
B-lymphocytes in the mantle-zone and T-lymphocytes in the
interfollicular areas and germinal centres (whereas the germinal
B-lymphocytes should be negative) and a strong staining of all the
follicular lymphomas.
87 laboratories
participated in the assessment, of which 53 achieved optimal
(61%), 28 good (32%), 4 borderline (5%) and 2 poor
marks (2%).
The
following antibody clones were used:
mAb clone 124
(DakoCytomation,
n=70)
mAb clone Bcl2/100/D5
(Ventana,
n=5; NovoCastra,
n=4; Medac,
n=1)
mAb clone 100
(BioGenex,
n=4)
mAb clone Bcl-2-100
(Zymed,
n=2)
mAb clone 8C8
(NeoMarkers,
n=1).
Optimal stains in
this assessment were obtained with the clones 124 (43/70),
Bcl2/100/D5 (6/10), 100 (3/4) and 8C8 (1/1).
All laboratories
achieving an optimal result used HIER. All of the heating buffers
Tris-EDTA/EGTA pH 9 (43/63), Citrate pH 6 – 7,2 (5/9), CC1 Ventana
Benchmark (1/7), EDTA pH 8 (2/5), TRS low pH 6.1 S1699
DakoCytomation (1/2) and TRS high pH 9.9 S3307 DakoCytomation (1/1)
could be used to obtain an optimal staining.
The mAb clone 124
was typically used in the range of 1:25 - 800 and resulted in an
optimal staining using one of the above mentioned HIER buffers. MAb
clone Bcl2/100/D5 was used in the range
of
1:20 – 100 with Tris-EDTA/EGTA
pH 9 or Citrate pH 6. MAb clone 100 was used in the range
of
1:300 –
500 with Tris-EDTA/EGTA pH 9 or Citrate pH 6, and mAb clone 8C8 was
used in 1:500 with Citrate pH 6.
The most frequent
cause of insufficient staining was:
- Too diluted primary antibody.
The prevalent
feature of an insufficient staining was
a too weak or completely
negative reaction in the tonsils and malignant lymphomas. Typically
there was no difference in the intensity in the normal and
neoplastic lymphocytes indicating that the protein expression in
these conditions is comparable, which enables the laboratories to
use normal tonsil as a reliable control for Bcl-2 in the
classification of lymphoma. In order to verify the sensitivity and
specificity the germinal centres are optimal. The isolated
T-lymphocytes in the germinal centres should be clearly demonstrated
while the germinal B-lymphocytes should be completely negative. |
