slide to be stained for CD20 comprised:
1. Appendix, 2. Tonsil, 3. Follicular lymphoma, 4. Precursor-B-ALL
All tissues were fixed in 10% neutral buffered formalin.
Criteria for assessing a CD20 staining as optimal included:
A strong, predominantly membranous staining of the mantle zone
B-cells, the germinal centre B-cells and the interfollicular B-cells in the tonsil and the appendix.
A strong membranous staining of virtually all the neoplastic cells
of the follicular lymphoma and the B-CLL.
A negative staining of the Precursor-B-ALL (only scattered maturated neoplastic
cells and entrapped normal B-cells may be demonstrated).
A negative staining of the
plasmacytoma (only the remnants of normal B-cells should be
A negative staining of all other cell types.
115 laboratories submitted stains. At the
assessment 66 achieved optimal marks (57 %), 34 good (30 %), 12
borderline (10 %) and 3 poor marks (3 %).
The following Abs were used:
mAb clone L26 (Dako, n=99, Ventana n=11, BioGenex n=1,
NeoMarkers n=1, Zymed n=1)
mAb clone 7D1 (Novocastra n=1)
pAb RN-9013 (NeoMarkers n=1)
Optimal staining for CD20 in this assessment
was obtained with the mAb clone L26 (65 out of 113) and the
pAb RN-9013 (1 out of 1).
L26: The protocols
giving an optimal result were all based on heat induced epitope
retrieval (HIER) using Tris-EDTA/EGTA pH 9 (31/59)*, Cell
Conditioning1 (BenchMark, Ventana) (15/24), Citrate pH 6 or 7
(7/11), EDTA/EGTA pH8 (3/4), Target Retrieval Solution pH 6,1 (TRS,
Dako) (3/3), Bond Epitope Retrieval Solution 2 (Bond, Vision
Biosystems) (2/4), Bond Epitope Retrieval Solution 1 (Bond, Vision
Biosystems)(1/1), 1mM EDTA pH 9 (1/1) or PT Module Buffer 1 pH 6 (LabVision)
(1/1) as HIER buffer. The mAb was typically diluted in the range of
1:50 – 1:3,000** depending on the total sensitivity of the protocol
employed, or as a Ready-To-Use (RTU) antibody. Using these protocol
settings 95 out of 108 (88 %) laboratories produced a sufficient
staining (optimal or good).
* (number of optimal results/number of
laboratories using this buffer)
** Dako has changed the anti-CD20 Ig concentration
recently, which explains the wide range of Ab concentration
protocol giving an optimal result was based on HIER using
Tris-EDTA/EGTA pH 9 and a dilution of 1:1.000 of the primary Ab.
The most frequent causes of insufficient
- Too low concentration of the primary antibody
- Omission of HIER.
The prevalent feature of the insufficient
results was a general too weak and diffuse staining of both the
normal and neoplastic cells supposed to be demonstrated, mainly related to a too low
concentration of the primary Ab resulting in an imprecise and patchy
staining of the membranes especially in the mantle zone B-cells and
the the B-CLL.
Tonsil is appropriate for control tissue: the staining of the B-cells
should be as strong as possible with no reaction in other cells (epithelial cells,
muscle cells etc.).
HIER is mandatory to obtain an optimal
reaction. Efficient HIER in an alkaline buffer and/or with the use
of a pressure cooker typically allowed the laboratories to use a
lower concentration of the clone L26 compared to a milder HIER in a Citrate
buffer pH 6.
However, the use of the milder HIER seemed to be beneficial to the morphology and resulted in a very crisp and
precise localization of CD20. The difference in HIER did not affect
CD20 was also assessed in run 6 2002, where 62
laboratories participated. Even though the number of participants
has almost doubled, the results of the 2 runs are comparable, as the proportion of sufficient stains
increased from 81% t o 87%.
The mAb clone L26 and the pAb RN-9013 are useful for
the demonstration of CD20. HIER is mandatory to obtain an optimal
result. Concentration of the primary Ab should be carefully
calibrated. Tonsil is an appropriate control: The
mantle zone B-cells and the germinal centre B-cells must show a
strong reaction. No other cells should stain.