slide to be stained for Immunoglobulin Kappa (IgK) comprised:
1. Chronic lymphatic lymphoma (CLL), IgK positive,
2. Mantle cell
lymphoma (MCL), IgL positive,
3. Tonsil fixed 4 h, 4. Tonsil fixed
24 h, 5. Tonsil fixed 72 h.
All specimens were fixed in 10 % NBF.
Criteria for assessing an IgK staining as optimal included:
A strong and distinct membranous staining of approximately half of
the normal B-cells in the mantle zone in the tonsils.
A strong cytoplasmic reaction of approximately half of the plasma cells.
A strong and distinct membranous reaction of the majority of the
neoplastic cells in the CLL.
- No staining of the neoplastic cells of the MCL.
- A weak general background staining only.
79 laboratories submitted stains, including one in situ
hybridization (ISH) stain for IgK mRNA, which was accepted as
equivalent to the immunohistochemical demonstration of IgK.
At the assessment 9 achieved optimal marks (11 %), 12 good (15 %), 15 borderline (19 %) and 43
(55 %) poor marks.
The following Abs were used:
mAb clone 6E1 (Immunotech, n=1)
mAb clone A8B5 (Dako, n=5)
mAb clone HP6053 (Zymed, n=1)
mAb clone KDB-1 (BioCare, n=1)
mAb clone MH19-1 (CLB, n=1)
mAb clone R10-21-F3 (Dako, n=5)
pAb 760-2514 (Ventana, n=2)
pAb A0191 (Dako, n=55)
pAb A0192 (Dako, n=2)
ISH 780-2843 (Ventana, n=1)
Optimal staining for IgK in this assessment was obtained with the
following Abs: pAb A0191 (8 out of 55) and pAb A0192 (1 out of 2).
All optimal protocols were based on HIER.
Using the pAb A0191 both Citrate pH 6.0 and Target Retrieval
Solution S1699 (TRS; Dako) could
be used as HIER buffer: 6 out of 19 and 2 out of 8 gave optimal
results, respectively. In the optimal protocols the pAb was
typically used in the range of 1:2.000 – 8.000 depending on the
sensitivity of the protocol applied.
Using the pAb A0192 (now discontinued from the vendor) with citrate pH 6.0 as the HIER buffer gave an
optimal result with an Ab dilution of 1:10.000.
The combination of pAb A0191 in a proper dilution (1:2.000 - 8.000)
and HIER in Citrate pH 6.0 or TRS gave an optimal staining in 9 out
of 21 laboratories (43 %).
The most frequent causes of insufficient staining were (often in
- Too low concentration of the primary antibody
- Too high concentration of the primary antibody
- Inappropriate epitope retrieval (proteolysis or HIER in an
- Less succesful primary Ab.
The prevalent feature of an insufficient staining was a too weak or
negative staining of the normal mantle zone B-cells and the CLL. The
membranes of the normal IgK positive B-cells should be distinctively
demonstrated with only a minimal background reaction in the mantle
zone. In almost all slides in which the normal IgK positive B-cells
were selectively demonstrated, the protocol could be used for the
demonstration of the IgK in the CLL (with no simultaneous positivity
in the mantle zone lymphoma).
Virtually all protocols demonstrated the plasma cells. However, the
cytoplasmic IgK expression in plasma cells is much stronger than
that of normal and neoplastic B-cells. Thus, plasma cells can not be
used as control for the demonstration of membranous IgK in
Another feature of the insufficient staining was over staining
decorating all mantle zone B-cells. This was most frequently due a
too high concentration of the primary Ab, which made it impossible
to differentiate between the membranous IgK reaction and an
intercellular background reaction of immunoglobulin. In these
protocols it was also impossible to demonstrate any certain
difference between the CLL and MCL.
Proteolytic pre-treatment could not be used to obtain an optimal
staining. Typically the membranes of normal B-cells as well as
neoplastic cells were digested causing a false negative reaction for
IgK while at the same time enhancing the intercellular background
reaction. In general the staining result using proteolytic digestion
is very dependent on the fixation time in NBF and might be optimised
to the individual specimen, but in a diagnostic setting the fixation
conditions are very comparable to the range of the applied fixation
times (4 – 72 hours) of the tonsils used in this assessment and the
procedure should optimally be applicable to various fixation times.
Using HIER in Citrate pH 6 or TRS an optimal reaction could be
obtained in all of the specimens in the multitissue block,
indicating that HIER is the preferable pre-treatment for IgK.
In this assessment, pAb A0191 (Dako)
was the most useful Ab for IgK. HIER in Citrate pH 6.0 or TRS was
the most appropriate pre-treatment.
The concentration of the primary Ab should be carefully calibrated. Normal tonsil is an appropriate
control tissue: approximately 50% of the mantle zone B-cells should
show a distinct membrane staining reaction, while the rest should be