 The
slides to be stained for
Epithelial antigen (EA) included an ovary (specimen
2; EA in
epithelial inclusion glands and oocytes), appendix
(specimen 3; EA in mucosal enterocytes), kidney (specimen 4; EA in collecting tubules and patchy in Bowman's
capsule) and adrenal cortex
(specimen 6; no EA), a testis/epididymis with malignant mesothelioma
(specimen 1; EA in epididymis epithelium), and a lung
adenocarcinoma (specimen 5; EA in tumour cells).48
laboratories submitted stainings. All used mAb Ber-EP4 from
DakoCytomation. At the
assessment, 17 were deemed optimal, 20 acceptable, 8 borderline
and 3 poor (see left diagram). The basis for an
optimal result was an intense and distinct staining of cell
membranes - particularly basolateral - and less pronounced diffuse
cytoplasmatic staining of cells expected to stain. |
 |
|
The best
pre-treatment appeared to be
HIER in Target Retrieval Solution (TRS;
S1699 or S1700, DakoCytomation). 13 out of 18 laboratories which
obtained an optimal result used this pre-treatment. Antigen
retrieval could also be obtained with
proteolytic pre-treatment
(Proteinase K or Protease type XXIV, Sigma). However, an optimal
result was seen in only 3 out of 26 laboratories (see
right diagram).
The most
frequent parameters giving suboptimal reactions were:
-
insufficient HIER (citrate buffer pH 6), and
- inadequate proteolytic digestion.
This was
most evident in the lung carcinoma,
whereas in the
appendix the
enterocytes stained well in almost all protocols. This emphasises the
need for a careful selection of control
tissue, e.g.,
kidney (confer Fig. 1a).
|
|
|
|
Examples of good protocols |
|
|
|
 |
 |
|
Fig. 1a. Optimal staining
of kidney using mAb Ber-EP4. The
collecting renal tubules show intense staining
with basolateral memebrane enhancement.
Also some cells of the Bowman's capsule are stained. The proximal
tubules are unstained. |
Fig.
1b. Acceptable staining.
Same field and Ab as in Fig. 1a.
The collecting renal
tubules show
weaker staining
than in Fig. 1a, and the Bowman's capsule is unstained. |
 |
 |
|
Fig.
1c. Insufficient staining. Same field and Ab as in Fig. 1a. The
collecting tubules are unstained. False positive staining of
erythrocytes (unsuppressed pseudoperoxidase). |
Fig. 1d. Insufficient staining. Same field and
Ab as in Fig. 1a. The tubules (particularly the proximal) show false positivety due to
endogenous biotin. |
 |
 |
|
Fig.
2a.
Optimal staining of lung adenocarcinoma using mAb Ber-EP4. Same
protocol as in Fig. 1a. Intense staining of all tumour cells.
|
Fig.
2b.
Acceptable staining of lung adenocarcinoma. Same
field and Ab as in Fig. 1a. Same protocol as in Fig. 1b.
|
 |
 |
|
Fig.
2c.
Insufficient staining of lung adenocarcinoma.
Same field and Ab as in Fig. 2a. Same protocol as in Fig. 1c.
The tumour cells are weakly stained or unstained. |
Fig. 2d. Insufficient staining of lung
adenocarcinoma. Same field and Ab as in Fig. 2a. Same protocol
as in Fig. 1d. The epithelial antigen of the tumour cells is
unstained, but a weak false positive staining due
to endogenous biotin is seen as a granular product in the
cytoplasm. |
