slide to be stained for membranous
Lymphatic Leukaemia (B-CLL), IgK positive, 2. B-CLL, IgL positive,
Tonsil fixed 4 hours,
4. Tonsil fixed 72 hours, 5. Tonsil fixed
All specimens were fixed in 10 % NBF.
Criteria for assessing a membranous IgK staining as optimal
strong and distinct membranous staining of approximately half
of the normal peripheral B-cells in the mantle zones in the tonsils.
strong and distinct membranous reaction of the majority of the
neoplastic cells in the CLL of IgK subtype (verified by IHC
and flow cytometry).
staining of the neoplastic cells of the B-CLL of IgL subtype
(verified by IHC and flow cytometry).
strong cytoplasmic reaction in approximately half of the plasma
cells and the immunoblasts in the germinal centres.
weak background reaction was accepted, as long as the interpretation
was not compromised.
laboratories participated in the assessment. 11
achieved optimal marks (14 %), 22 good (27 %), 17 borderline (21 %)
and 30 (38 %) poor marks.
following Abs were used:
mAb clone R10-21-F3 (Dako, n=4)
mAb clone A8B5 (Dako, n=2)
mAb clone HP6053 (Zymed, n=1)
mAb clone kp-53 (Novocastra, n=1)
mAb clone L1C1 (Biogenex, n=1)
pAb A0191 (Dako, n=61)
pAb A0192 (Dako, n=6)
pAb N1510 (Dako, n=1)
pAb 760-2514 (Ventana, n=2)
pAb RB-333 (Neomarkers n=1)
Optimal staining for IgK in this assessment was only obtained with
the pAb A0191 (11 out of 61).
All 11 optimal protocols were based on heat induced epitope retrieval (HIER) using either
citrate pH 6.0, Target
Retrieval Solution pH 6.1 (Dako TRS, S1699/1700) or citraconic anhydride pH 6.0 as the heating buffer. The pAb
A0191 was typically used in range of 1:3,000 - 1:12,000
depending on the total sensitivity of the protocol employed.
With the pAb A0191 after HIER in one of
the above mentioned buffers and a Ab dilution in the range of
1:3,000 – 12,000, 27 laboratories out of 38 obtained an sufficient
mark (72 %), 11 (29 %) were marked as optimal. The most
robust and reproducible procedure was based on HIER in TRS pH 6.1 as
11 laboratories out of 14 (78 %) obtained a sufficient mark, whereas
using citrate pH 6.0 15 out of 23 laboratories (65 %) had a
The most frequent causes of insufficient staining were:
- Less successful primary antibody
- Too low concentration of the primary antibody
- Too high concentration of the primary antibody
- Inappropriate epitope retrieval (proteolytic
Almost all laboratories were able to demonstrate
the IgK 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 IgK in the mantle zone
lymphocytes and the B-CLL of IgK subtype. A too weak or false negative staining
was seen in 89 % of the insufficient results (43 out of 47).
11 % (4 out of 47) a too strong staining was observed giving a
false positive staining of IgK in the B-CLL of IgL subtype.
In all the insufficient results due to weak or false negative staining
this feature was seen in both the two B-CLLs and in all three tonsil specimens.
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 IgM.
IgK was also assessed in
run 15. In that run 79 laboratories
participated out of which 74 % (58 laboratories) obtained an
insufficient mark. Each laboratory was given a specific
recommendation to improve their protocol. 49 laboratories, which
obtained insufficient results in run 15, submitted a new IgK stain
in run 18. 22 of the laboratories followed the recommendation and 11
of these improved to either good or optimal marks (50
%). 27 laboratories did not follow the recommendation. Only 2 of these
(14 %) obtained a sufficient staining in run 18.
In this assessment, the pAb A0191 (Dako) was the most useful
Ab for the demonstration of membranous IgK. HIER in citrate pH 6.0,
Target Retrieval Solution pH 6.1 or citraconic anhydride was the
most appropriate pre-treatment methods.
The concentration of the primary Ab should be carefully calibrated.
Normal tonsil is an appropriate control tissue: approximately half of the peripheral mantle zone B-cells should show a distinct
membranous staining reaction for IgK, while the remaining mantle
zone B-cells (which are IgL producing) should be unstained.
Fig. 1a. Optimal staining for IgK of
the tonsil. Even at a low magnification x10 a proportion of the
B-cells in the mantle zone of the secondary follicles are
demonstrated (compare with Fig. 2a). The protocol was based on the pAb A0191 correctly
calibrated and HIER in TRS pH 6.1.
Fig. 1b. Insufficient staining for IgK
of the tonsil, same field as in Fig. 1a. At the low magnification x10 the B-cells in
zone of the secondary follicles are negative and only the plasma
cells are identified (compare with Fig. 2b). The protocol was based on HIER in
6.0 and the pAb A0191 but in a too low concentration.
Fig. 2a. High magnification x40 of the optimal staining of a secondary follicle in the tonsil.
Approximately 50–60 % of the mantle zone B-cells show a distinct
membranous reaction and only a minimal background reaction.
Fig. 2b. High magnification x40 of the insufficient staining of a secondary follicle in the
tonsil, same field as in Fig. 2a. Only the plasma cells and immunoblasts are positive, while
all the mantle zone B-cells are false negative. Same protocol as in