slide to be stained for
1. Skin, 2. Appendix, 3. Breast, 4. Malignant melanoma.
All tissues were fixed in 10% neutral buffered formalin.
Criteria for assessing a S100 staining as optimal included:
- A strong, distinct nuclear and
cytoplasmic staining reaction of the normal melanocytes, the
Langerhans cells and (when present) the myoepithelial cells of the
sweat glands in the skin. No staining should be seen in
the squamous epithelial cells.
- A strong, distinct nuclear and
cytoplasmic staining reaction of the macrophages in lamina propria,
the Schwann cells and satellite cells in the muscularis propria
and submucosa in the appendix. The
epithelial cells and muscle cells should be negative.
- A moderate to strong, distinct nuclear
and cytoplasmic staining of the myoepithelial cells in the
breast, and no more than a focal reaction in the epithelial cells.
- A strong, distinct nuclear and cytoplasmic
staining of the majority of the neoplastic cells of the metastatic
- A strong, distinct nuclear and cytoplasmic reaction
of fat cells and macrophages in all specimens.
106 laboratories submitted stains. At the
assessment, 35 obtained an optimal result (33%), 44 good (42%), 25
borderline (24%) and 2 poor (2%).
The following Abs were used:
mAb clone 4C4.9 (Ventana, n=2; NeoMarkers, n=1)
mAb clones 15E2E2 (BioGenex, n=1; DCS, n=1)
pAb 18-0046 (Zymed, n=1)
pAb 760-2523 (Ventana, n=3)
pAb MS-296-P (NeoMarkers, n=1)
pAb RB-9081-P (NeoMarkers, n=1)
pAb Z0311 (Dako, n=95)
Mandatory for an optimal result in this
assessment was the use of pAb Z0311 (35 out of 96
laboratories; 37%) using following protocol settings:
Both Heat Induced Epitope Retrieval (HIER) and enzymatic
pre-treatment. Using HIER 24 out of 57 laboratories obtained an optimal
result (42%), using enzymatic pre-treatment 10 out of 28
laboratories obtained an optimal result (36 %). Among 11
laboratories omitting pre-treatment, one obtained an optimal result (9%).
With HIER 17 laboratories used Tris-EDTA/EGTA pH 9, 3 Ventana Cell
Conditioning 1, 2 citrate buffer pH 6.0 and one Dako TRS-buffer
With enzymatic pre-treatment 4 laboratories used Ventana protease 1, 4 used
Dako proteinase K and 2 used protease type XXIV.
The pAb Z0311 was typically diluted in the range of 1:800 – 1:8,000
depending on the total sensitivity of the protocol employed.
Using these protocol settings 75 out of 90 laboratories (83%)
produced a sufficient staining (optimal or good).
The most frequent causes of insufficient
- Too low concentration of the primary antibody
- Less successful primary antibody
- Excessive enzymatic pre-treatment
- Omission of epitope retrieval.
Virtually all laboratories
were able to demonstrate S-100 in the malignant melanoma, whereas
the prevalent feature of an insufficient staining was a general too
weak or false negative staining of the normal melanocytes,
the Langerhans cells and the myoepithelial cells. HIER and enzymatic
pre-treatment seemed nearly equally useful to obtain an optimal
result. However, differences in the staining patterns were seen with
these two pre-treatment methods: Using HIER, the melanoma cells were
uniformly stained, while using enzymatic pre-treatment the staining
showed a more heterogeneous pattern and in several cases the
cytoplasmic compartment was totally extracted due to an excessive
With HIER and pAb Z0311 50 out of 57 (88%)
laboratories obtained a sufficient result, while 19 out of 28
laboratories (68%) using enzymatic pre-treatment and pAb Z0311 obtained a
Omission of epitope retrieval typically resulted
in a general too weak reaction and 6 of 11 laboratories using this
procedure received an insufficient mark.
S-100 was also assessed in Run 7 2003, in which 63 laboratories
The overall proportion of sufficient staining increased from 71 % in
run 7 to 75% in the present run (however, the number of participants
doubled from run 7 to run 20, so for many of the laboratories, this
was the first time to stain for S-100). The proportion of poor results
decreased from 13% in run 7 to 2% in run
20 while borderline staining increased from 16% to 24%.
Skin seemed to be a good and
useful control for S-100, as all laboratories with optimal staining
of the other tissues could demonstrate melanocytes and
The pAb Z0311 appears to be a robust and good marker for
S-100. HIER should be the preferred
pre-treatment. Skin is an appropriate control: the melanocytes and
the Langerhans cells must show a strong and distinct reaction, while
the squamous epithelial cells shall remain negative.
Insufficient results seem in many laboratories to be due to lack
of awareness of optimal positive and negative control material
giving difficulties in finding the right pre-treatment and dilution
of the primary Ab.