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Recommended S100 protocols  ■  Recommended S100 control tissue

Assessment Run 34 2012
 

S100
The slide to be stained for S100 comprised:

1. Skin, 2. Appendix, 3. Breast hyperplasia, 4. Malignant melanoma,
5. Schwannoma

All tissues were fixed in 10% neutral buffered formalin.

Criteria for assessing an 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 of the peripheral nerve fibres and the ganglionic satellite cells in the muscularis propria and submucosa in the appendix. The epithelial cells and muscle cells should be negative.
  • An at least weak but distinct nuclear and cytoplasmic staining reaction of the follicular dendritic cells in the germinal centres of the Peyer’s plaques in the appendix.
  • A moderate to strong, distinct nuclear and cytoplasmic staining reaction 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 reaction of the majority of the neoplastic cells of the  melanoma and the schwannoma.
  • A strong, distinct nuclear and cytoplasmic staining reaction of the fat cells and macrophages in all specimens.

200 laboratories participated in this assessment. 64 % achieved a sufficient mark. In table 1 the antibodies (Abs) used and marks are summarized.

Table 1. Abs and assessment marks for S100, run 34

Concentrated Abs

N

 Vendor Optimal Good Borderl. Poor Suff.1 Suff. OPS2
mAb clone 4C4.9 1
1
1		 Master Diagnostica
Thermo/NeoMarkers
Zytomed		 0 2 0 1 - -
mAb clone 12E2E2 1 Biogenex 0 0 1 0 - -
mAb clone cocktail
12E2E2+4C4.9		 1 Biocare 0 1 0 0 - -
rmAb clone EP32 2 Epitomics 0 2 0 0 - -
pAb Z0311 131 Dako 36 46 35 14 63 % 80 %
pAb NCL-S100p 5 Leica/Novocastra 0 3 2 0 60 % -
pAb RB-9018-p 3 Thermo/NeoMarkers 0 3 0 0 - -
pAb E031 1 Linaris 0 0 0 1 - -
Ready-To-Use Abs                
mAb clone 4C4.9
330M-17		 1 Cell Marque 0 0 0 1 - -
mAb clone 4C4.9
MON-RTU1191		 1 Monosan 0 0 0 1 - -
mAb clone 4C4.9
790-2914		 12 Ventana 0 5 4 3 42 % -
mAb clone cocktail
12E2E2+4C4.9
PM089		 1 Biocare 0 0 0 1 - -
pAb IR/IS504 24 Dako 0 23 1 0 96 % -
pAb 760-2523 10 Ventana/Cell Marque 0 2 7 1 20 % -
pAb clone PA0900 3 Leica 0 3 0 0 - -
pAb 1 Unknown 0 1 0 0 - -
Total 200   36 91 50 23 -  
Proportion     18 % 46 % 25 % 11 % 64 %  

1) Proportion of sufficient stains (optimal or good), 2) Proportion of sufficient stains with optimal protocol settings only, see below.

The following central protocol parameters were used to obtain an optimal staining:

Concentrated Abs
pAb Z0311: The protocols giving an optimal result were all based on heat induced epitope retrieval (HIER) using either Cell Conditioning 1 (CC1, BenchMark, Ventana) (12/40)*, Target Retrieval Solution pH 9 (3-in-1) (TRS pH 9 (3-in-1), Dako)(9/18/)*, TRS pH 9 (Dako) (5/11)*, Bond Epitope Retrieval Solution 2 (BERS 2, Bond, Leica) (4/9)*, Bond Epitope Retrieval Solution 1 (BERS 1, Bond, Leica) (1/1)*, Tris-EDTA/EGTA pH 9 (3/6)* or Citrate pH 6 (2/6)* as the retrieval buffer.
The mAb was typically diluted in the range of 1:750-1:10.000 depending on the total sensitivity of the protocol employed. Using these protocol settings 67 out of 84 (80 %) laboratories produced a sufficient staining (optimal or good).
* (number of optimal results/number of laboratories using this buffer)

The most frequent causes of insufficient staining were:
- Too low concentration of the primary antibody
- Less successful primary antibody
- Proteolytic pre-treatment and no pre-treatment

In this assessment and in concordance with the observations in the previous assessment of S100 (run 20, 2007), the prevalent feature of an insufficient staining was a too weak or completely false negative reaction in cells expected to be demonstrated. A too weak or false negative staining was seen in 88 % of the insufficient results (64 out of 73). Virtually all the participating laboratories were able to demonstrate S100 in the peripheral macrophages, the Schwann cells and the neoplastic cells of the schwannoma, whereas the demonstration of S100 in the myoepithelial cells, the Langerhans cells and in particular the follicular dendritic cells of the germinal centres was much more challenging and was only seen with appropriate protocol settings, e.g., a correct titre of the pAb Z0311, Dako and the use of HIER. Omission of epitope retrieval gave a significantly reduced sensitivity: In 18 out of 25 protocols without epitope retrieval but otherwise optimal protocol settings an insufficient result was obtained and none were assessed as optimal.
Virtually the same insufficient results were seen when proteolytic pre-treatment was used: 15 out of 23 protocols gave an insufficient result and none were optimal. The use of proteolysis both caused a reduced sensitivity and an impaired morphology as in several cases the cytoplasmic compartment of many cells e.g., the neoplastic cells of the melanoma and the epithelial cells of the breast was totally extracted due to an excessive digestion.

In this run only the pAb Z0311, Dako applied with HIER and diluted in the range of 1:750 - 10.000 of the concentrated format could be used to obtain an optimal staining reaction for S100 in all the tissues. Using these protocol settings, the overall pass rate was 80 %. For the Ready-To-Use (RTU) product of the same pAb IR/IS504, Dako, a pass rate of 100 % was seen if same protocol settings were applied. However, none were assessed as optimal, primarily due to a slightly reduced sensitivity compared to the staining obtained by the concentrated format.

Appendix was found to the most reliable and informative control for S100. In the optimal protocols an at least weak to moderate but distinct nuclear and cytoplasmic staining reaction in the follicular dendritic cells of the Peyer’s plaques were seen indicating that these cells might be used as critical staining quality indicator for S100. The Schwann cells must be stained as strongly as possible, but still without any staining reaction of the smooth muscle cells and the epithelial cells.
In this context it may be superior to use tonsil (in stead of appendix) as positive control for S100, as the number of lymphoid follicles in tonsillar tissue is more constant. Prompt and adequate fixation in formalin is crucial to stabilize and preserve S100, especially in tissue components with a relatively low level of the S100 antigen such as the follicular dendritic cells.

This was the 3rd assessment of S100 in NordiQC (Table 2). A decrease in the pass rate was seen from both run 7, 2003, and run 20, 2007.
 

Table 2: Proportion of sufficient results for S100 in the three NordiQC runs performed

  Run 7 2003 Run 20 2007 Run 34 2012

Participants, n=

 63 106 200

Sufficient results

 71 % 75 % 64 %


The lower pass rate may be due to several factors: New tissue material circulated and many new participants. The laboratories participating for the first time obtained a significantly lower pass rate compared to the laboratories also participating in the previous run 22, 2007: For the laboratories participating for the first time the pass rate was 57 % (59 out of 102), whereas the pass rate was 71 % (68 out of 106 laboratories) for the laboratories participating in both runs.

Conclusion
In this assessment the pAb Z0311 was the only antibody giving optimal staining results for S100. HIER and an appropriate titre of the primary antibody were mandatory for an optimal performance. Omission of epitope retrieval or proteolytic pre-treatment gave a significantly inferior result.
Tonsil is recommendable as positive control: The follicular dendritic cells must show an at least weak to moderate distinct nuclear and cytoplasmic staining reaction. Alternatively appendix may be used: A strong staining reaction must be seen in the Scwann cells, while no staining reaction should be seen in the smooth muscle cells
Fig. 1a. Optimal S00 staining of the appendix using the pAb Z0311 with HIER in an alkaline buffer. The peripheral nerves and macrophages in lamina propria show a strong cytoplasmic and nuclear staining reaction and a weak to moderate staining reaction is seen in the follicular dendritic cells in the germinal centre. No background staining is seen. Also compare with Figs. 2a – 4a, same protocol. Fig. 1b. Insufficient S100 staining of the appendix using the pAb Z0311 by protocol settings providing a too low sensitivity – primarily due to a low concentration of the primary Ab., same field as in Fig. 1a.
Virtually no staining reaction is seen in the follicular dendritic cells in the germinal centre and the staining reaction in the macrophages and peripheral nerves is significantly reduced – also compare with Figs. 2b - 4b, same protocol.
Fig. 2a. Optimal S100 staining of the appendix using the same protocol as in Fig. 1a. The Schwann cells and the satellite cells of the peripheral nerves show a moderate to strong staining reaction. No staining is seen in the smooth muscle cells. Fig. 2b. S100 staining of the appendix using same protocol as in Fig. 1b., same field as in Fig. 2a. The proportion and intensity of the positive cells is reduced. However still a weak to moderate staining reaction is seen, indicating that peripheral nerves can not reliably be used as positive control for S100 – also compare with Figs. 3b - 4b same protocol.
Fig. 3a. Optimal S100 staining of the breast hyperplasia using same protocol as in Figs. 1a & 2a. The myoepithelial cells show a moderate to strong cytoplasmic and nuclear staining reaction. A weaker staining of the secretory cells is seen, but no background staining is seen. Fig. 3b. Insufficient S100 staining of the breast hyperplasia using same protocol as in Figs. 1b & 2b., same field as in Fig. 3a. The myoepithelial cells show a weak and equivocal staining reaction also compare with Fig. 4b, same protocol.
Fig. 4a. Optimal S100 staining of the malignant melanoma using same protocol as in Figs. 1a - 3a. Virtually all the neoplastic cells show a moderate to strong cytoplasmic and nuclear staining reaction. Fig. 4b. Insufficient S100 staining of the malignant melanoma using same protocol as in Figs. 1b – 3b., same field as in Fig. 4a. The neoplastic cells show a weak and equivocal staining reaction.
Fig. 5a. Insufficient S100 staining of the breast hyperplasia using proteolytic pre-treatment. The cytoplasmic compartment of both the myoepithelial cells and the glandular epithelial cells is digested and only the moderately stained nuclei are left. Also compare with Fig. 5b, same protocol. Fig. 5b. Insufficient S100 staining of the malignant melanoma using proteolytic pre-treatment, same protocol as in Fig. 5a. The cytoplasmic compartment is digested  and only the moderately stained nuclei are left.
 
SN/MV/LE

Last update 29-03-2012