 The
slide to be stained for CD30
comprised:
1. Tonsil, 2. Embryonal carcinoma, 3-4. Hodgkin lymphoma classical
type, NS.
All tissues were fixed in 10 % neutral buffered formalin.
Criteria for assessing a CD30 staining as optimal included:
- An at least moderate and distinct
membranous staining of the activated interfollicular and
perifollicular B- & T-cells in the tonsil.
- An at least weak to moderate, distinct
membranous and dot-like cytoplasmic staining of the majority of
the Hodgkin cells in the two Hodgkin lymphomas.
- A moderate to strong, distinct membranous
staining of majority of the neoplastic cells in the embryonal
carcinoma.
- A strong cytoplasmic staining in the
plasma cells in all specimens.
- No or only a minimal background reaction.
126 laboratories participated in the
assessment. 78 % achieved a sufficient mark. The results are summarized in Table 1.
Table 1.
Abs and scores for CD30, run 25
|
Concentrated Abs |
N |
Vendor |
Optimal |
Good |
Borderl. |
Poor |
Suff.1 |
Suff. OPS2 |
|
mAb clone
Ber-H2 |
98
3
1 |
Dako
NeoMarkers
Zymed |
47 |
32 |
21 |
2 |
78 % |
81 % |
|
mAb clone
1G12 |
3 |
Novocastra |
2 |
1 |
0 |
0 |
- |
- |
|
mAb clone
15B3 |
2 |
Novocastra |
1 |
1 |
0 |
0 |
- |
- |
|
mAb clone
HRS4 |
1 |
NeoMarkers |
0 |
1 |
0 |
0 |
- |
- |
|
Ready-To-Use Abs |
|
|
|
|
|
|
|
|
|
mAb clone
Ber-H2 |
12 |
Ventana |
3 |
6 |
2 |
1 |
75 % |
86 % |
|
mAb clone
Ber-H2 |
6 |
Dako |
0 |
4 |
2 |
0 |
67 % |
- |
|
Total |
126 |
|
53 |
45 |
25 |
3 |
- |
- |
|
Proportion |
|
|
42 % |
36 % |
20 % |
2 % |
78 % |
83 % |
1) Proportion of sufficient stains
(optimal or good), 2) Proportion of sufficient stains with optimal
protocol settings only, see below.
The following central protocol parameters were used to obtain an
optimal staining:
Concentrated Abs
mAb clone Ber-H2: The protocols giving an optimal result were
all based on heat induced epitope retrieval (HIER) using
Tris-EDTA/EGTA pH 9 (22/45)*, Cell Conditioning 1 (BenchMark,
Ventana) (6/16), Target Retrieval Solution pH 9 (EnVision FLEX TRS high pH,
Dako, (5/10)*, Bond Epitope Retrieval Solution 2 (Bond, Leica)
(5/8), Target Retrieval Solution pH 6.1 (Dako) (4/6), Bond Epitope
Retrieval Solution 1 (Bond, Leica)(2/3), Citrate pH 6 (2/7) or EDTA/EGTA
pH 8 (1/3) as retrieval buffer. The mAb was typically diluted in the
range of 1:10 – 1:300 depending on the total sensitivity of the
protocol employed. Using these protocol settings 79 out of 97 (81 %)
laboratories produced a sufficient staining (optimal or good).
* (number of optimal results/number of
laboratories using this buffer)
mAb clone 1G12: The protocols giving an optimal result were
all based on heat induced epitope retrieval (HIER) using Bond
Epitope Retrieval Solution 2 (Bond, Leica)(1/2)* or Tris-EDTA/EGTA
pH 9 (1/1)*. The mAb was diluted in the range of 1:10 – 1:40
depending on the total sensitivity of the protocol employed. Using
these protocol settings 3 out of 3 (100 %) laboratories produced a
sufficient staining.
mAb clone 15B3: The protocol giving an optimal result was
based on heat induced epitope retrieval (HIER) using Cell
Conditioning 1 (BenchMark, Ventana). The Ab was diluted 1:30. Using
similar protocol settings 2 out of 2 (100 %) laboratories produced a
sufficient staining.
Ready-To-Use Abs
mAb clone Ber-H2, prod. no. 760-2926, Ventana: The
protocols giving an optimal result were based on HIER in Cell
Conditioning 1, standard, an incubation time of 32-48 min in the
primary Ab and UltraView or iView as the detection system. Two
laboratories used amplification kit. Using these protocol settings 6
out of 7 (86 %) laboratories produced a sufficient staining.
The most frequent causes of insufficient staining were:
- Too low concentration of the primary antibody
- Insufficient HIER – too short heating time or use of citrate pH
6.0
- Proteolytic pre-treatment
In this assessment the prevalent feature of the insufficient results
was a false negative or too weak staining in the cells expected to
be demonstrated. This was especially observed in the Hodgkin
lymphoma no. 4 and the embryonal carcinoma, whereas the staining was
acceptable in the Hodgkin lymphoma no. 3 in the majority of the
stains submitted.
Normal tonsil was found to be a reliable positive control for CD30,
provided that the activated inter- and perifollicular B- & T-cells
showed a distinct membranous staining and focally also a dot-like
reaction. If these cells were negative or only weakly demonstrated,
the neoplastic cells in the Hodgkin lymphomas and embryonal
carcinoma also were negative or only showed an equivocal reaction.
Occasionally a weak reaction was seen in serum in the vessels,
especially when a highly sensitive protocol was applied (e.g., a
3-step polymer based detection system combined with HIER in an
alkaline buffer). This was fully acceptable, as this reaction did
not interfere with the interpretation of CD30. Proteolytic
pre-treatment could not be used to obtain a sufficient result, as
both the number of positive cells was reduced compared to the result
based on HIER, but also due to an impaired morphology, as the
membranes of the cells were extracted by the enzymatic digestion.
This was the second assessment of CD30 in NordiQC, and the
proportion of sufficient results declined from 92 % in run 11, 2004
to 78 % in the current run. The lower pass rate is probably due to
more challenging tissue material circulated.
Table 2:
Sufficient results with CD30 in two runs
| |
Run 11
2004 |
Run 25 2009 |
|
Participants, n= |
74 |
126 |
|
Sufficient results |
92 % |
78 % |
Conclusion
The mAbs clones BER-H2, 1G12 and 15B3 are all useful markers for
CD30.
HIER - preferable in an alkaline buffer - is mandatory to have an
optimal reaction for CD30.
Tonsil is recommended as positive control: The activated
interfollicular and perifollicular B- & T-cells shall show a
moderate to strong and distinct membranous reaction. |
