slide to be stained for
Tonsil fixed 4 h,
3: Tonsil fixed 48 h,
fixed 96 h,
5-7: Nodal T-cell lymphomas.
Criteria for assessing a
staining as optimal included:
- A strong and distinct predominantly
membranous reaction of the majority of neoplastic cells in the 3 T-cell
- A strong and distinct predominantly
membranous staining of all normal T-cells in the tonsils and liver
- No staining in other cells.
Especially B-cells in the tonsil should be negative
87 laboratories participated in the assessment.
29 achieved optimal staining (33 %), 35 good (40 %), 20 borderline
(23 %) and 3 (5 %) poor staining.
The following Abs were used:
mAb clone F7.2.38 (DakoCytomation n=33)
mAb clone PS1 (Novocastra n=20, Ventana n=12)
mAb clone SP7 (NeoMarkers n=7)
mAb clone UCHL1 (DakoCytomation n=1)
pAb A0452 (DakoCytomation n=11)
pAb NCL-CD3p (Novocastra n=2)
pAb RB-360 (NeoMarkers n=1)
Optimal staining in this assessment was
obtained with following Abs: clone F7.2.38 (11 out of 33 were
optimal), clone PS1 (10 out of 32 were optimal), clone (rabbit)
SP7 (2 out of 7 were optimal), pAb A0452 (5 out of 11
were optimal), and pAb RB-360 (1 out of 1 was optimal).
All optimal protocols were based on HIER
irrespectively of the Ab employed.
With clone F7.2.38 only Tris-EDTA/EGTA
pH 9 was used as HIER buffer in optimal protocols. F7.2.38 diluted
1:50 – 1:100 combined with HIER in Tris-EDTA/EGTA pH 9 resulted in
an optimal staining in 11 out of 23 laboratories.
With clone PS1 the following HIER buffers were used in
optimal protocols: Tris-EDTA/EGTA pH 9 (8 out of 16 were optimal),
EDTA pH 8 (1 out of 3 was optimal) and Citrate pH 6 (1 out of 2 was
optimal). This Ab could also be used as a Ready-To-Use product.
Using a concentrated Ab diluted 1:50-100, 8 out of 18 obtained an
optimal staining, while only 2 out of 12 using a Ready-To-Use Ab
obtained an optimal staining.
With clone SP7 only Tris-EDTA/EGTA pH 9 could be used as HIER
buffer (2 out of 3 were optimal). For an optimal result, SP7 was
either diluted 1:100 using a water bath or 1:800 using a pressure
cooker as heating device for HIER.
With pAb A0452 the following buffers were used for an optimal
result: Tris-EDTA/EGTA pH 9 (4 out of 9 were optimal) and EDTA/EFTA
pH 8 (1 out of 2 was optimal). The pAb was typically diluted in the
range of 1:300 – 1:400.
pAb RB-360 was used with HIER in Tris-EDTA/EGTA pH 9 and
diluted 1:200 giving an optimal result.
The most frequent causes of insufficient stains
were (often in combination):
- Too low concentration of the primary antibody
- Too high concentration of the primary antibody
- False positive reaction due to endogenous biotin
- Insufficient as well excessive epitope
- Apparently inappropriate choice of primary Ab
The prevalent feature of an insufficient staining was a too weak
staining or false negative staining of the T-cells. Also a false
positive reaction due to endogenous biotin was observed as a common
feature, primarily in liver cells and appendiceal columnar cells.
This was typically seen in staining combining efficient HIER in an
alkaline buffer as Tris-EDTA/EGTA pH 9 or EDTA pH 8 and a biotin
based detection system.
The T-cells in the tonsils should stain as intensely as possible without reaction of the B-cells. A good quality indicator
was the germinal centres, in which the isolated T-cells should be
distinctively labelled with no reaction of the B-cells. In general using a biotin based detection system
without biotin blocking, HIER in Citrate pH 6 is preferable to
Tris-EDTA/EGTA pH 9 to minimize the appearance of endogenous biotin.
A higher concentration of the primary Ab should be used to
compensate for the lower sensitivity.
CD3 was also assessed in run 5. In that run 45 laboratories
participated, out of which 14 (31 %) were assessed as insufficient,
close to the present proportion (28%). However, many laboratories
participated in the CD3 assessment for the first time.