The slides to be stained
for Melan-A comprised:
1: malignant melanoma
(small intestine), 2: granulosa cell tumour (ovary),
malignant melanoma (testis), 4: adrenal gland, 5: blue
Criteria for assessing a Melan-A staining as optimal
included: a strong and
distinct intracytoplasmic reaction in the normal
melanocytes and the tumour cells of
the malignant melanoma and the blue naevus.
If mAb A103 was used, also a distinct staining of the
granulosa cell tumour and adrenal cortical cells
should be seen. A weak non-specific staining of enterocytes
is accepted. All other cells should remain unstained.
laboratories submitted stainings. At the assessment, 14
laboratories achieved optimal staining
(40%), 10 good (29%), 8 borderline
(23%), and 3
poor staining (8%).
33 used mAb
used mAb Melan A Ab-3 (Neomarkers), a cocktail
of the two clones M2-7C10
and M2-9E3. Optimal results could be
obtained with both mAb A103 and the melan-A Ab3 cocktail.
Using mAb A103, it
was mandatory to use HIER in Tris-EDTA/EGTA pH 9 to obtain
an optimal staining. While melanocytes and the malignant melanoma could in
most cases be stained with less
sensitive protocols, the granulosa cell tumour and the adrenal cortex could
dilution for A103 ranged from 1:25 to 1:200.
Using the mAb cocktail M2-7C10
and M2-9E3 an optimal result was obtained with HIER in Citrate pH 6 (one
The most frequent causes of insufficient
stainings (often in combination) were:
- A too
low concentration of the
- No or insufficient HIER
(citrate pH 6 or too short heating time with Tris-EDTA/EGTA