slide to be stained for membranous
Lymphatic Leukaemia (B-CLL),
Tonsil fixed 4 hours, 4. Tonsil fixed 72 hours, 5. Tonsil fixed
All specimens were fixed in 10 % NBF.
Criteria for assessing a membranous IgM staining as optimal
strong distinct membranous staining of the majority of the mantle zone B-cells in the germinal centres of the tonsils.
Distinct membraneous/cytoplasmic staining of activated B-cells, centroblasts and centrocytes
in the germinal centres of the tonsils.
distinct membranous staining of the majority of the neoplastic cells
in the two B-CLLs.
strong cytoplasmic reaction in
all plasma cells and immunoblasts.
weak background reaction was accepted, as long as the interpretation
was not compromised.
laboratories participated in the assessment. 13
achieved optimal marks (21 %), 6 good (10 %), 12 borderline (20 %)
and 30 (49 %) poor marks.
The following Abs were used:
mAb clone R1/69 (Dako n=5)
mAb clone 1D7-F10 (BioGenex n=1)
pAb 414-01 (Signet n=1)
pAb 760-2654 (Ventana n=3)
pAb A0091 (Dako n=2)
pAb A0425 (Dako n=39)
pAb A0426 (Dako n=6)
pAb N1509 (Dako n=1)
pAb NCL-IgMp (Novocastra n=2)
pAb RB1434 (NeoMarkers n=1)
Optimal staining for IgM in this assessment was only obtained with
the pAb A0425 (13 out of 39 were optimal).
All the optimal protocols were based on heat induced epitope retrieval (HIER).
As HIER buffer both Target Retrieval Solution pH 6.1 (S1699
Dako), Tris-EDTA/EGTA pH 9, Citrate pH 6.0 and Cell
Conditioning 1 (CC1, Ventana) could be used to obtain an optimal
In the optimal protocols the pAb A0425 was used in the range
of 1:200 – 1:1,000 depending of the total sensitivity of the protocol
With pAb A0425 after HIER in
one of the above mentioned buffers and a primary Ab dilution between
1:200 – 1:1,000, 16 out of 19 obtained an sufficient mark
(84 %), of which 13 (68 %) were optimal.
The most frequent causes of insufficient staining were:
- Less successful primary antibody
- Too low concentration of the primary antibody
- Omission of epitope retrieval
- Proteolytic pre-treatment
In the assessment almost all laboratories were able to demonstrate
the IgM in the cytoplasm of the plasma cells and the
immunoblasts in the germinal centres, whereas the prevalent
feature of the insufficient staining was a too weak or false
negative staining of the membranous IgM of the neoplastic B-cells in
the two B-CLL. A too weak or false negative staining was seen in
95 % of the insufficient results (40 out of 42) and in only 5 % (2
out of 42) a too strong staining was observed.
In all the insufficient results, weak or false negative staining of the two B-CLLs
were seen in parallel with weak or false negative staining in
all three tonsil specimens. If the staining was assessed as too
weak, the mantle zone B-cells also showed a too weak membranous
staining and if the staining was too strong the background staining
in the interfollicular areas and the squamous epithelium showed a
too strong and a false positive staining, respectively.
Normal tonsil seems to a reliable positive control in which
virtually all the peripheral mantle zone B-cells shall show a strong
distinct membranous reaction with a minimal background reaction in
the interfollicular areas (only circulating peripheral B-cells and
plasma cells should be demonstrated in these areas).
In most stains (also when optimal) a non-specific
background reaction was observed in the tonsil fixed for 4 hours,
whereas in the tonsils fixed for 72 and 168 hours the signal-to-noise ratio was improved and the identification of the IgK positive
B-cells facilitated, indicating that a short fixation time
in NBF may impede the interpretation of the IgK reactivity. The same
feature was noted with IgK.
In this assessment, pAb A0425 (Dako) was the most useful Ab
for the demonstration of membranous IgM. HIER was the only appropriate pre-treatment. The concentration of the primary Ab
should be carefully calibrated. Normal tonsil is an appropriate
control tissue in which the mantle zone B-cells should show a
distinct membranous staining with only a minimal background