|
 The
slide to be stained for CD23
comprised:
1-3. Tonsils fixed 4h, 72h, and 168h, resp., 4-5. Chronic
lymphatic leukaemia (B-CLL), 6. Mantle cell lymphoma (MCL).
All tissues were fixed in 10% neutral buffered formalin.
Criteria for assessing a CD23 staining as optimal included:
-
A strong, distinct membranous staining of
the activated B-cells in the mantle zone of the germinal centres
in the tonsils.
-
A strong, distinct staining of the
follicular dendritic cells in the germinal centres.
-
A strong, distinct membranous staining of
majority of the neoplastic cells in the two B-CLLs.
-
No
staining of MCL.
88 laboratories submitted stains. At the
assessment 25 achieved optimal marks (28 %), 23 good (26 %), 29
borderline (33 %) and 11 poor marks (13 %).
The following Abs were used:
mAb clone 1B12 (Novocastra, n=56; Ventana, n=5; Cell Marque,
n=3; Monosan, n=2; NeoMarkers, n=2)
mAb clone BU38 (The Binding Site, n=2)
mAb clone MHM6 (Dako, n=10)
rmAb SP23 (NeoMarkers, n=8)
Optimal staining for CD23 in this assessment
was obtained with the mAb clone 1B12 (21 out of 68; 31%) and the
rmAb clone SP23 (4 out of 8; 50%).
1B12: The protocols giving optimal results were
all based on heat induced epitope retrieval (HIER) using an alkaline
buffer as Tris-EDTA/EGTA pH 9, Bond Epitope Retrieval Solution 2
(Bond, Vision Biosystems) or Cell Conditioning1 (BenchMark,
Ventana). The mAb was typically diluted in the range of 1:20 – 1:200
depending on the total sensitivity of the protocol employed. Using
these settings 39 out of 51 laboratories (76 %) produced a
sufficient staining (optimal or good).
Only 1 of 9 protocols based on the use of 1B12 as a
Ready-To-Use (RTU) Ab gave a sufficient staining result, despite the protocol
settings being otherwise identical to the those based on a concentrated
Ab giving sufficient results.
SP23:
The protocols giving an optimal result were all based on HIER using
Tris-EDTA/EGTA pH 9 as buffer. The Ab was diluted in the range of
1:100 – 1:200 depending on the total sensitivity of the protocol
employed. Using these settings all of 7 laboratories produced a
sufficient staining.
The most frequent causes of insufficient
staining were:
- Less successful primary Ab
- Less successful RTU Ab clone 1B12
- Too low concentration of the primary Ab
- HIER in non-alkaline buffer (typically Citrate pH 6.0)
In the assessment virtually all laboratories were able to
demonstrate CD23 in the follicular dendritic network within the
germinal centres, whereas the prevalent feature of an insufficient
staining was a too weak or false negative staining of the mantle
zone B-cells and neoplastic B-cells in the two B-CLLs. In general,
CD23 is only weakly expressed in B-CLL and a sensitive
protocol is required to demonstrate CD23 properly.
Normal tonsil was found to be a reliable
quality indicator for the immunohistochemical demonstration of CD23,
as the sufficient results all showed a distinct membranous reaction
of the activated mantle zone B-cells. If these B-cells were negative
or only weakly demonstrated, the B-CLLs also were negative or only equivocally
stained.
CD23 was also assessed in
Run 9 2003,
in which 57 laboratories participated.
The overall proportion of sufficient staining decreased from 76 % in
run 9 to 55 % in the present run. This is probably due to several
factors including many new participants, more challenging tissue
material circulated, and a higher proportion of the laboratories
using 1B12 as an RTU product.
Including only the laboratories participating in both runs (n=56)
the proportion of sufficient staining decreased from 75 % 66 %.
13 laboratories, which obtained an insufficient result in run 9,
submitted a new CD23 stain in run 19. 10 of them followed the
recommendations and 7 improved their result to good or optimal (70
%). 3 laboratories did not follow the recommendations and 1 of these
(33 %) obtained a sufficient staining in run 19.
The choice of Ab clone and format (concentrated v. RTU) had a
high impact on the final assessment result. Only the concentrated
format of clones 1B12 and SP23 (i.e., the dilution is calibrated by
the laboratories) could give optimal results.
It is particularly noteworthy that none of 10 laboratories using
clone MHM6 obtained an optimal result in this run (in run 9, 2 out of
8 was optimal).
Conclusion
The mAb clones 1B12 and SP23 are useful
for the detection of CD23 in B-cell lymphomas. HIER in an alkaline
buffer (Tris-EDTA/EGTA pH 9, BERS2 or CC1) is mandatory for optimal
performance.
The current RTU
Abs have not
been calibrated properly to fulfil the diagnostic demands. The Ab
concentration in the RTU format must be related to the total
sensitivity of the protocol employed, and this sensitivity has to be
defined in a clinical setting and validated by the producer as well
as the laboratories. A tonsil/lymph
node is appropriate control: The activated mantle zone B-cells must
show a distinct membranous reaction. It has to be emphasized that
the follicular dendritic cells shall not be used as positive control
for CD23, as these cells strongly express CD23 and can consequently
not be used to calibrate the immunohistochemical protocol. |
