The slide to be stained for membranous
immunoglobulin M (IgM) comprised:
1. Mantle cell lymphoma, 2. B-Chronic lymphatic leukaemia (B-CLL),
3. Tonsil fixed 24 hours, 4. Tonsil fixed 48 hours, 5. Tonsil fixed
All specimens were fixed in 10 % neutral buffered formalin.
Criteria for assessing a membranous IgM staining as optimal included:
- A strong distinct membranous staining of virtually all the mantle
zone B-cells of the germinal centres of the tonsils.
- A distinct membranous staining of the majority of the neoplastic
cells in the mantle cell lymphoma and the B-CLL.
- A strong cytoplasmic reaction in plasma cells, immunoblasts and the
follicular dendritic network in the germinal centres of the tonsils.
A weak background reaction was accepted, as long as the
interpretation was not compromised.
80 laboratories submitted stains. At the assessment 15 achieved
optimal marks (19 %), 25 good (31 %), 19 borderline (24 %) and 21
poor marks (26 %).
The following Abs were used:
mAb clone R1/69 (Dako, n=2)
mAb clone IgM88 (BioGenex, n=1)
mAb clone RVS-M (Chemicon, n=1)
pAb 760-2654 (Ventana, n=4)
pAb A0425 (Dako, n=58)
pAb A0426 (Dako, n=5)
pAb N1509 (Dako, n=2)
pAb A0091 (Dako, n=1)
pAb P0215 (Dako, n=1)
pAb P0322 (Dako, n=1)
pAb NCL-IgMp (Novocastra, n=3)
pAb RB-1434 (NeoMarkers, n=1)
Optimal staining for IgM in this assessment was only obtained with
the pAb A0425 (15 out of 58, 26%).
The 15 optimal protocols were based on heat induced epitope
retrieval (HIER) - except for one laboratory which used a combination
of proteolytic pre-treatment (Proteinase K) and HIER (Citrate pH 6.0). The HIER buffers used
in the 14 protocols were Citrate pH 6.0 (5/22)*,
Target Retrieval Solution pH 6.1 (Dako TRS, S1699/1700) (7/10)*,
Bond Epitope Retrieval Solution 1 (Bond, Leica Microsystems) (1/2)*,
Cell Conditioning1 (BenchMark, Ventana) (1/4)* and TRIS-EDTA/EDTA pH
The pAb A0425 was typically used in the range of 1:300 -
1:5.000 depending on the total sensitivity of the protocol employed. Using these protocol settings 32 out of 43 laboratories (74%)
produced a sufficient staining assessed as good or optimal.
* (number of optimal results/number of laboratories using this
The most frequent causes of insufficient staining were:
- Less successful primary antibody
- Too low concentration of the primary antibody
- Inappropriate epitope retrieval (proteolytic pre-treatment or no
In this assessment the prevalent feature of an insufficient staining
was a too weak or false negative staining of the membranous IgM of
the neoplastic cells in the two B-cell lymphomas and the normal
mantle zone B-cells, whereas virtually all could demonstrate the
cytoplasmic IgM in plasma cells. A too weak or false negative
staining was seen in 98 % of the insufficient results (39 out of 40),
which typically displayed by a diffuse or dot-like
cytoplasmic reaction with no accentuation of the membranes of the
neoplastic cells in the lymphomas.
None of 10 protocols based on proteolytic
pre-treatment gave a sufficient result (with
these protocols only the cytoplasmic IgM in plasma cells and
demonstrated, with no membranous reaction in the lymphatic B-cells
(probably due to digestion of the membrane epitopes on these cells).
IgM was also assessed in
Run 18 2006, in which 61 laboratories
The overall proportion of sufficient staining has increased from 31
% in run 18 to 50 % in the present run. In the assessment of IgM run
18, each of 42 laboratories, which obtained an insufficient result, was
given a specific recommendation to improve their protocol. 35 of
these laboratories submitted a new IgM stain in run 23. 20 followed
the recommendations, of which 14 improved to good or optimal (70 %).
13 laboratories did not follow the recommendation, and 1 of these (8
%) obtained a sufficient staining in run 23. Three laboratories changed
their entire IHC platform and one improved.
There are several reasons for this constantly low proportion of
sufficient results and the limited impact of specific
recommendations to the laboratories with insufficient results.
First, continuous use of less successful Abs, in particular mAbs, which in none of the
two runs have
given an optimal result - irrespective of the protocols
applied; second, usage of proteolytic pre-treatment for
the pAb A0425 (Dako) instead of HIER; and third, many new
laboratories participating in an IgM assessment for the first time. Though it cannot be excluded that other Abs than
pAb A0425 would be able give optimal IgK stains with better
datasheets provided with these Abs have apparently not provided any
good solutions. Some laboratories only use anti-IgM for plasma
cells and plasma cell disorders. However, the material provided from
NordiQC needs an IgM protocol aimed at demonstrating membranous IgM,
not cytoplasmic, in order to diagnose lymphomas.
In concordance with the previous IgM assessment, 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). There was not noticed any significant
difference in the reaction pattern in the tonsils fixed in the range
of 24 – 72 hours in 10 % NBF, when optimal protocol settings (HIER)
In this assessment, pAb A0425 (Dako) was the most useful Ab for the
demonstration of membranous IgM. HIER was the most robust
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 reaction.