 The
slide to be stained for Cyclin D1 (CyD1)
comprised:
1. Tonsil fixed 48 h, 2. B-Cell chronic
lymphatic leukaemia (B-CLL) 3 - 4.
Mantle cell lymphoma.
All specimens were fixed in 10 % NBF.
Criteria for assessing a CyD1 staining as optimal included:
- A moderate to strong, distinct nuclear
staining of the suprabasal squamous epithelial cells in the
tonsil.
- A moderate to strong, distinct nuclear
reaction of the neoplastic cells in the two mantle cell lymphoma.
- No nuclear reaction in the
neoplastic cells of the B-CLL.
In all specimens a nuclear reaction in
endothelial cells and a weak cytoplasmic reaction was accepted.
87 laboratories submitted stains. At the assessment 30 achieved optimal marks (35 %), 21
good (24 %), 14 borderline (16 %) and 22 (25 %) poor marks.
The following Abs were used:
rmAb clone SP4 (NeoMarkers, n=50; Ventana, n=2; Cell Marque,
n=2; DCS, n=1)
mAb clone P2D11F11 (Novocastra, n=12; Ventana, n=3; Vector, n=1)
mAb clone DCS6 (Dako, n=9; Ventana, n=1; Calbiochem, n=1)
pAb CP236 (Biocare, n=5)
Optimal staining for CyD1 in this assessment
was obtained with the rmAb clone SP4 (29 out of 54) and
the pAb CP236 (1 out of 5). All optimal protocols were based on
heat
induced epitope retrieval (HIER).
With clone SP4 all protocols
resulting in an optimal staining were based on HIER using one
of the following buffers:
Tris-EDTA/EGTA pH 9 – 21/34 using this obtained an optimal
mark,
CC1 (Cell Conditioning 1, Ventana) – 3/8 using this obtained
an optimal mark,
TRS pH 6,1 (Dako) – 2/4 using this obtained an optimal mark,
Citrate pH 6 – 2/4 using this obtained an optimal mark,
EDTA pH 8 – 1/2 using this obtained an optimal mark.
Clone SP4
typically was used in the range of 1:25 – 1:200 depending on the total
sensitivity of the protocol employed. SP4 could also be used
as an RTU Ab. The combination of the clone SP4, properly diluted, and HIER in Tris-EDTA/EGTA pH 9 or CC1
resulted in an optimal staining in 24 out of 43 laboratories (56 %)
and an sufficient staining - optimal or good - in 40 out of 42
laboratories (95 %).
With pAb CP236 the protocol
resulting in an optimal stain was based on HIER using
Tris-EDTA/EGTA pH 9 as the heating buffer, the Ab diluted
1:100. CP236 typically gave a weak cross reactivity of the
membranes of epithelial cells. This was accepted, as it did not
interfere with the interpretation.
The most frequent causes of insufficient staining were:
- Less successful primary antibody
- Too low concentration of the primary
antibody
- Insufficient heat induced epitope
retrieval
In this assessment the prevalent feature of an
insufficient staining was a too weak or completely false negative
nuclear staining of the neoplastic cells in the two mantle cell
lymphoma and frequently the false negative reaction was accompanied
by a moderate to strong false positive cytoplasmic reaction in both
the B-CLL and mantle cell lymphoma.
Normal tonsil can be used as both positive and negative control to
validate the sensitivity and specificity of the protocol for CyD1.
In the squamous epithelium, the suprabasal cells should display a
strong nuclear reaction with only minimal cytoplasmic
reaction. In the lymphatic areas only the endothelial cells should
show a nuclear reaction.
It should also be noticed that the most
frequent cause for insufficient staining in this assessment seemed
to be related to the primary Ab. Thus, 10 out of 11
laboratories (95 %) using the mAb clone DCS6 obtained an insufficient
mark. Likewise, 14 out of 16 laboratories (88 %) using the mAb
clone P2D11F11 were marked as insufficient. All were using HIER
and comparable protocols as applied for the clone mAb SP4 and
the pAb CP236.
CyD1 was also assessed in
Run 9 2003, in which
57 laboratories participated. Out of these 27 laboratories (47 %)
had an insufficient staining. Each laboratory was given a specific
recommendation how to improve their protocol. 25 laboratories, which
obtained an insufficient result in Run 9
submitted a new CyD1 stain in Run 17. 17 out of these followed the
recommendation, and 11 (65 %) improved their mark from insufficient
to either good or optimal. 8 laboratories did not follow the
recommendations and one (13 %) improved from insufficient
to optimal.
The overall proportion of insufficient
staining was reduced from 47 % in run 9 to 41 % in the present run.
Focusing only on the laboratories participating in both runs (n=54)
the proportion of insufficient staining was more significantly
reduced from 48 % (n=26) to 37 % (n=20).
The results of the CyD1 assessment in
Run 17 is in accordance with the results and conclusions of
the study based on the NordiQC results of Run
9: Antibody Selection in Immunohistochemical Detection of Cyclin D1
in Mantle Cell Lymphoma by Emina Torlakovic et al., Am J Clin Pathol
2005;124:782-789:
“It is apparent from the results of NordiQC testing and the results
of this study that many laboratories use suboptimal protocols and
suboptimal antibody selection. Both problems might be identified by
participation of the laboratories in external quality control
programs”. Conclusion
The rmAb clones SP4 and the pAb CP236 are useful Abs
for CyD1.
HIER, preferably in an alkaline buffer as Tris-EDTA/EGTA pH 9, is
mandatory for optimal performance.
Tonsil is an appropriate control for CyD1
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