slide to be stained for Synaptophysin
1. Brain, 2. Appendix, 3. Colon adenocarcinoma, 4. Pancreas blood
type A, 5. Pancreas blood type 0, 6-7. Endocrine lung carcinomas, 8.
Medullary thyroid carcinoma.
All tissues were fixed in 10% neutral buffered formalin.
Criteria for assessing a SYP staining as optimal
- A strong and distinct cytoplasmic reaction of the normal
neuroendocrine cells in the appendiceal mucosa and in the islet cell
in the pancreas (a weak background reaction around the islets was
accepted due to diffusion of the protein in the two pancreas
- At least a moderate, distinct granular cytoplasmic reaction of
the normal ganglion cells and axons in the appendiceal Aurbach’s
plexus and in the brain.
- At least a moderate, distinct cytoplasmic reaction in the majority
of the neoplastic cells of the two endocrine lungcarcinomas.
- A strong and distinct cytoplasmic
reaction of the medullary thyroid carcinoma.
- Scattered positive cells in the colon adenocarcinoma –
while the large majority of tumour cells should be negative.
113 laboratories submitted stains. One laboratory used an
inappropriate antibody. At the assessment of 112 laboratories 20
achieved optimal marks (18 %), 45 good (40 %), 37 borderline (33 %)
and 10 poor marks (9 %).
The following Abs were used:
mAb clone 27G12 (Novocastra, n=22)
mAb clone Snp88 (BioGenex, n=16)
mAb clone SY38 (Dako, n=10; BioGenex, n=1)
rmAb clone SP11 (NeoMarkers, n=6)
pAb A0010 (Dako, n=39)
pAb N1566 (Dako, n=3)
pAb 760-2668 (Ventana, n=10)
pAb 18-0130 (Zymed, n=3)
pAb NCL-SYNAPp (Novocastra, n=1)
pAb RB-1461 (NeoMarkers, n=1)
Optimal staining for SYP in this assessment was obtained with the
mAb clone 27G12 (11 out of 22), the mAb clone Snp88 (6 out of 16),
the pAb A0010 (1 out of 39) and the pAb 18-130 (2 out of 3).
27G12: The protocols giving an optimal result
were all based on heat induced epitope retrieval (HIER) using Tris-EDTA/EGTA pH 9 (7/11)*, Citrate pH 6 (2/2) or Target Retrieval
Solution pH 9 (FLEX TRS high pH, Dako) (1/1). The mAb was typically
diluted in the range of 1:30 – 1:300 depending on the total
sensitivity of the protocol employed. Using these protocol settings
14 out of 14 (100 %) laboratories produced a sufficient staining
(optimal or good).
* (number of optimal results/number of laboratories using this
Snp88: The protocols giving an optimal result
were all based on HIER using Tris-EDTA/EGTA pH 9 (4/10) or Bond Epitope Retrieval Solution 2
(Bond, Leica Microsystems; 2/2). The mAb was diluted in the range
of 1:100 – 1:3,000 depending on the total sensitivity of the
protocol employed. Using these protocol settings 11 out of 11 (100
%) laboratories produced a sufficient staining.
18-0130: The protocols giving an optimal result were
based on heat induced epitope retrieval (HIER) using Tris-EDTA/EGTA
pH 9 (1/1) or no retrieval (1/1). The mAb was diluted in the range
of 1:50 – 1:200 depending on the total sensitivity of the protocol
employed. Using these protocol settings 2 out of 2 (100 %)
laboratories produced a sufficient staining (optimal or good).
A0010: The protocol giving an optimal result was
based on HIER using Tris-EDTA/EGTA pH 9 (1/28). The pAb was used in
the dilution 1:100. Using these protocol settings 5 out of 7 (71 %)
laboratories produced a sufficient staining.
The most frequent causes of insufficient staining
- Less successful primary antibody
- Too low concentration of the primary antibody
The prevalent features of the insufficient
results were 1) A generally too weak or false negative
reaction (66 %), 2) An excessive background reaction (11 %), or 3) A combination of
a weak reaction and excessive background reaction giving a poor
signal-to-noise ratio (23 %).
One of the central parameters for a sufficient SYP demonstration
was the choice of the primary antibody.
In this assessment the mAb clone SY38 gave an insufficient reaction in 11 out of 11 protocols. The
SY38 typically obtained an acceptable reaction in the normal endocrine
cells and peripheral nerves in the appendix, but a false negative
reaction in large proportions of the neoplastic cells in the
endocrine lung carcinomas, sometimes also in the medullary thyroid
The false positive reactions were typically observed when the 2 pAbs
A0010 (Dako) and 760-2668 (Ventana) were used. The pAb
A0010 mainly gave a general background reaction e.g a cytoplasmic
cross reaction in the appendiceal enterocytes and of unexplained
reasons also a strong nuclear reaction in both the pancreas and the
The pAb 760-2688 typically gave a non-specific dot-like cytoplasmic
reaction in several cell types as the appendiceal enterocytes, the acinar
pancreatic cells, and the colon adenocarcinoma. Laboratories trying
to reduce this non-specific reaction by diluting the antibody
obtained too weak
reactions of the neuroendocrine tumours.
In the multitissue block used for the assessment in this run two pancreas
specimens from patients
with blood group 0 and blood group A, respectively. In stains with
mAb clone Snp88, a distinct dot-like cytoplasmic reaction of the
acinar cells in the pancreas from the patient with blood group A was
seen. This pattern, which has been described in the literature as
Golgi" (MAG), is undesired and the
interpretation using such Abs has to be taken with care, as the
pattern highly mimics the SYP reaction in endocrine
tumours. NordiQC will contact the producer of the ascites mAb
suggesting a change to a supernatant Ab to reduce this source of error.
The overall pass rates for the most used
Abs for SYP in the latest two assessments are shown in the table:
Run 18 2006
Run 22 2008
mAb clone 27G12
mAb clone Snp88
mAb clone SY38
These data clearly indicates the most robust markers for SYP
are the clones 27G12 and Snp88.
From the assessment it can be difficult to identify a robust and
reliable control for SYP. Appendix was in run 18 found to a
recommendable control, in which the peripheral nerves should display a
moderate to strong reaction. However, it appears that mAb clone SY38
can give a moderate to strong staining of the
peripheral nerves and still leave the endocrine carcinomas too weak
or even false negative.
At present the best recommendation is still to use appendix as
control but defer from using mAb SY38, and to calibrate the primary Ab to give a strong
reaction in the peripheral nerves in the Auerbach’s
and Meissner’s plexus as well as the axons in lamina propria beneath the epithelial cells.
Muscle cells must remain negative, whereas scattered epithelial
cells may be positive.
Compared to the assessment of SYP in
run 18 2006, an decrease in the
proportion of sufficient results from 69 % to 58 % was seen. This
decrease may be due to many new participants and the continuous use of less
25 laboratories, which obtained an insufficient result in run
18, submitted a new SYP stain in run 22. 14 of these followed the
recommendation given and 9 improved their marks to good or
optimal (64 %). 11 laboratories did not follow the recommendation,
and only 1 of these (9 %) obtained a sufficient staining in run 22.
In this assessment, the mAb clones 27G12 and Snp88 were the most
robust Abs for the demonstration of SYP. However, mAb clone Snp88 used
in an ascites format can give an undesired cross reactivity in
The concentration of the primary Ab should be carefully calibrated.
Normal appendix seems to be the most recommendable control tissue:
the axons of all the peripheral nerves in both the muscularis
and lamina propria must show a strong distinct granular reaction,
while the smooth muscle cells being negative.