 The
slide to be stained for MSA comprised:
1. Skin, 2. Kidney, 3-4. Malignant melanomas.
All tissues were fixed in 10% neutral buffered formalin.
Criteria for assessing a MSA staining as optimal included:
- A strong, distinct cytoplasmic staining of
virtually all the neoplastic cells of the malignant melanoma no. 3
and ≥ 50 % of the neoplastic cells of the malignant melanoma no.
4.
- No reaction in the kidney.
- No reaction in the skin with the exception
of scattered
reactive melanocytes.
102 laboratories submitted stains. Of these 3
used an inappropriate antibody. Assessment of the
remaining 99 laboratories gave following results: 50 achieved
optimal marks (51 %), 46 good (46 %), and 3 borderline (3 %). None of
the laboratories got poor marks.
The clone HMB-45 was used of all the
laboratories (Dako, n=85; NeoMarkers, n=4; Enzo, n=3; BioGenex, n=2; Novocastra, n=2; Ventana, n=2; Cell
Marque, n=1).
An optimal staining for MSA with clone
HMB-45 could in this assessment both be obtained without
pre-treatment (5 out of 8 labs) or by
using HIER (heat induced epitope retrieval) as pre-treatment (45 out of 87).
The HIER buffers used were:
Tris-EDTA/EGTA pH 9 (27/48)*,
Cell Conditioning1 (Benchmark, Ventana) (4/16),
Citrate pH 6 (6/9),
Target retrieval Solution pH 6.1 (Dako) (3/3),
EDTA pH 8 (1/3),
Bond Epitope Retrieval Solution 2 (Bond, Vision Biosystems) (2/2),
1mM EDTA, pH 9.0 (1/1).
* (number of optimal results/number of
laboratories using this buffer)
The mAb was typically diluted in the range of
1:25 – 1:400 depending on the total sensitivity of the protocol
employed or as a Ready-To-Use antibody. Using these settings 91 out
of 93 (98 %) laboratories produced a sufficient staining (optimal or
good). Proteolytic pre-treatment could not be used to obtain an
optimal staining and 2 out of 4 were insufficient.
The causes of insufficient staining were:
- Too low concentration of the primary antibody
- Proteolytic pre-treatment
96 out of 99 laboratories obtained a
sufficient result. The multitissue block consisted of two metastatic
melanomas with different MSA expression. The insufficient results were all
characterized by a too weak staining reaction in one of the melanomas.
Both HIER and omission of pre-treatment could in this assessment be used to obtain an
optimal staining and no major difference was seen in the pass rate
comparing these two protocol settings. However, in the previous
run 7 2003,
the performance without HIER was lower, and from a general point of
view HIER should be included in a standard protocol setting for MSA
to reduce any masking of the antigen by prolonged formalin fixation.
Omission of HIER may, however, be beneficial in cases of
problems with tissue detachment. Both procedures should be
validated by the individual laboratories in order to obtain the same
sensitivity in the two protocols (i.e., different
concentrations of the primary Ab may be necessary).
The proportion of sufficient MSA stains has increased from 74% in
run 7 to 97% in the current run, indicating that with a proper
adjustment of the protocol, clone HMB-45 is a robust Ab.
As normal melanocytes do not express MSA, melanocytic tumour tissue
is needed for control . The
staining of the tumour cells must be as strong as possible without
staining in other cells. A multitissue block combining
junction/compound nevi and malignant melanomas
with varying expression of MSA should be considered.
Conclusion
The mAb clone HMB45 is a robust Ab for the detection of MSA. HIER is
recommended for pretreatment even though omission of pre-treatment
also gives good results. |
