slide to be stained for ALK
1. Kidney, 2. Tonsil fixed 72 h, 3.
Anaplastic large cell lymphoma, 4. Diffuse large cell B-lymphoma.
All specimens were fixed in 10 % NBF.
Criteria for assessing an ALK staining as optimal included:
- A moderate to strong distinct predominantly
nuclear but also cytoplasmic staining of the majority of the
neoplastic cells of the anaplastic large cell lymphoma.
- The other tissues should be negative.
66 laboratories participated in the
assessment. Unfortunately 12 laboratories received slides from
NordiQC with only a limited number or total absence of neoplastic
cells in the anaplastic large cell lymphoma, which reduced the
number of participants to 54. At the assessment 40 of these achieved optimal
marks (74 %), 10 good (19 %), 2 borderline (4 %) and 2 (4 %) poor
The following Abs were used:
mAb clone ALK1 (Dako, n=44)
mAb clone 5A4 (Novocastra, n=3; NeoMarkers, n=1)
mAb clone ALK-01 (Ventana, n=3)
mAb clone SP8 (NeoMarkers, n=2)
pAb PS084 (Sanbio, n=1)
Optimal staining for ALK in this assessment was obtained with the
mAb clone ALK1 (35 out of 44), the mAb clone 5A4 (4
out of 4) and the mAb clone ALK-01 (1 out of 3).
All optimal protocols were based on Heat Induced Epitope Retrieval
Using the mAb clone ALK1 all protocols
resulting in an optimal staining were based on HIER using one
of the following buffers:
Tris-EDTA/EGTA pH 9 – 29/31 using this obtained an optimal
Citrate pH 6 – 3/8 using this obtained an optimal mark,
CC1 (Cell Conditioning 1, Ventana) - 1/2 using this obtained
an optimal mark,
TRS pH 6,1 (Target Retrieval Solution, Dako) - 1/1 using this
obtained an optimal mark.
The mAb clone ALK1 was typically used
in the range of 1:25 – 1:100 depending of the sensitivity of the
applied detection system.
Using the mAb clone 5A4 the protocols
resulting in an optimal result were all based on HIER using
Tris-EDTA/EGTA pH 9 and a dilution of 1:100 of the mAb. 4 out
of 4 using this setting obtained an optimal result.
The protocol resulting in an optimal result
using the mAb clone ALK-01 was based on HIER in CC1 and a RTU
The most frequent causes of insufficient
- Too low concentration of the primary
antibody or/and HIER in citrate pH 6
- Less successful primary antibody
In the assessment the prevalent feature of an
insufficient staining was a too weak staining of the neoplastic
cells in the anaplastic large cell lymphoma. The overall very high
pass rate of the laboratories for the ALK assessment is very
encouraging and will not be repeated in the immediate future.
Retrospectively more positive tumours should have been included in
the composed multitisue block to have a more subtle evaluation and
interpretation of the results.
As control for ALK only an anaplastic large
cell lymphoma seems to be a recommendable control, as no normal
tissue express the ALK protein.
The mAb clones ALK1, 5A4 and ALK-01 seem all to
be sensitive and reproducible markers for ALK. HIER preferably in an
alkaline buffer as Tris-EDTA/EGTA pH 9 or CC1 is recommended for