slide to be stained for Placental Alkaline Phosphatase (PLAP)
1-2. Testis with intratubular germinal cell neoplasia (IGCN), 3.
4. Embryonal carcinoma, 5. Placenta, 6. Appendix.
All tissues were fixed in 10% neutral buffered formalin.
Criteria for assessing a PLAP staining as optimal included:
- A strong and distinct predominantly membranous but also cytoplasmic
reaction of the majority of the neoplastic cells of the two IGCNs,
the seminoma and the embryonal carcinoma.
- A strong predominantly membranous but also cytoplasmic staining of
the cytotrophoblastic and syncytiotrophoblastic cells in the
placenta with no or only minimal reaction in the stromal cells.
- Negative staining of all other cells.
115 laboratories submitted stains. At the assessment 3 achieved
optimal marks (3 %), 57 good (50 %), 41 borderline (36 %) and 14
poor marks (12 %).
The following Abs were used:
mAb clone PL8-F6 (BioGenex, n=36)
mAb clone 8A9 (Dako, n=44; Novocastra/Leica, n=8)
mAb clone NB10 (Ventana, n=12)
mAb clone 8B6 (Dako, n=1)
rmAb clone SP15 (NeoMarkers/Thermo, n=8)
pAb A0268 (Dako, n=6)
Optimal staining for PLAP in this assessment was only obtained with
the mAb PL8-F6 (3 out of 36).
PL8-F6: All three optimal protocols were based on heat induced epitope
retrieval (HIER) using an alkaline buffer: Tris-EDTA/EGTA (2/21)*
or Bond Epitope Retrieval Solution 2 (Bond, Leica)
The mAb was diluted in the range of 1:100 – 1:800 depending
on the total sensitivity of the protocol employed. Using these
protocol settings 19 out of 22 (86 %) laboratories produced a
sufficient staining (optimal or good).
* (number of optimal results/number of laboratories using this
The low proportion of optimal results using the mAb clone PL8-F6
(8%) compared to the relative high pass rate of 86% of laboratories
using the Ab was due to the Mouse Ascites Golgi (MAG) reaction observed with the format as an ascites Ab of this clone. For yet
unexplained reasons the MAG reaction was not seen with the lot
MU2280600 used by 3 laboratories, whereas it was observed in all other lots. The other protocol settings (HIER, Ab titer and
detection system) used by these 3 laboratories were similar to the
laboratories getting the MAG reaction.
NordiQC has contacted BioGenex to identify the reason for the different reaction
patterns but not got an answer yet.
The most frequent causes of insufficient staining were:
- Too low concentration of the primary Ab
- Omission of HIER
- Less successful Abs (7/8 protocols using SP15 were insufficient)
- Less successful RTU Ab clone NB10 (7/12 were
In this assessment the prevalent feature of an insufficient staining
was a too weak or completely negative reaction of the neoplastic
cells in the three different germinal cell tumours and in particular the
neoplastic cells of the IGCNs. It was also observed that the two
most popular Abs, clone 8A9 and PL8-F6 both gave an undesired cross
reactivity. The clone 8A9 gave a distinct cytoplasmic cross reaction with
smooth muscle cells in all the tissues included in the block for the
assessment, whereas the clone PL8-F6 gave a dot-like cytoplasmic
reaction (MAG) in the epithelial cells of the appendix (which was
obtained from a patient with blood group A:
http://www.nordiqc.org/Techniques/Primary_antibody.htm). The cross
reaction of the mAb clone 8A9 was also addressed in the last assessment,
and the laboratories seem to try to reduce the reaction by e.g.
decreasing the concentration of the Ab. but this also reduces the
sensitivity and only 35% (18/52) using 8A9 obtained a sufficient
It was also observed that none of the other Abs used could be used
to obtain an optimal result and especially the rmAB clone SP15 was
less successful as 7/8 laboratories using this Ab obtained an
insufficient mark. The vendor recommends no retrieval should be
performed for the Ab, but all protocols omitting retrieval gave an
insufficient result as the IGCN was negative or too weak. If HIER
was performed the signal-to-noise ratio was compromised and both a
false positive cytoplasmic reaction and nuclear reaction was seen in
mature normal spermatocytes.
Finally when a RTU Ab is used, it is vital that the protocol
settings for the IHC procedure is adjusted carefully and in this
assessment 7/12 laboratories used protocol settings giving a too low
This was the second assessment of PLAP, and the proportion of sufficient
results declined to 52% in this run compared to 63% in run 14,
2005. The lower pass rate is probably due to a combination
of an increase in the number of participants (from 77 in run 14 to 115 in this
run) and more challenging tissue material circulated.
It was difficult to use placenta as a recommendable control for
PLAP, as this mainly could give information about the sensitivity but
not about the specificity. Regarding the sensitivity the cytotrophoblastic and syncytiotrophoblastic cells in the placenta
should give an as strong as possible staining with minimal reaction
in the stromal cells and to evaluate any unspecific reaction
appendix, preferable blood type A, should be used.
The mAb clone PL8-F6 was the only Ab that could give an optimal
result. HIER in alkaline buffer was mandatory. Caution should be
taken when the Ab is used, as the ascites format of the Ab gives
cross reaction in blood type A tissue. Due to the many difficulties
with the used PLAP Abs, the laboratories should consider to use
other Abs such as OCT 3/4.