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Recommended ALK protocols  ■  Recommended ALK control tissue

Assessment Run 17 2006
 

Anaplastic lymphoma kinase (ALK)

The slide to be stained for ALK comprised: 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 marks.

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 (HIER).

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 mark,
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 Ab.

The most frequent causes of insufficient staining were:
- 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.

Conclusions
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 optimal performance.

Fig. 1. Staining for ALK of the anaplastic large cell lymphoma assessed as optimal. The majority of the neoplastic cells show a distinct nuclear and cytoplasmic reaction. No background reaction is seen.
Fig. 2. Staining for ALK of the anaplastic large cell lymphoma assessed as good. The neoplastic cells are demonstrated, but the intensity is weaker compared to the result obtained in Fig. 1. However the nuclear details are more pronounced.
Fig. 3. Insufficient staining for ALK in the anaplastic large cell lymphoma. The neoplastic cells are only weakly stained, complicating the interpretation.
MV/SN/AS

Last update 23-06-2006