The slide to be stained for CD20 comprised:

1. Appendix, 2. Tonsil, 3. Diffuse large B-cell lymphoma (DLBCL), 4. Precursor-B-Acute lymphatic leukaemia (Pre-B-ALL), 5. B-Chronic lymphatic leukaemia (B-CLL), 6. Plasmacytoma.

All tissues were fixed in 10% neutral buffered formalin.

Criteria for assessing a CD20 staining as optimal included:

• A strong, predominantly membranous staining reaction 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 reaction of virtually all the neoplastic cells of the DLBCL.
• A moderate to strong membranous staining reaction of virtually all the neoplastic cells of the B-CLL.
• No staining of the Pre-B-ALL (only scattered maturated neoplastic cells and entrapped normal B-cells may be demonstrated).
• No staining of the plasmacytoma (only residual normal B-cells should be demonstrated).
• No staining of all other cell types.

167 laboratories participated in this assessment. 95 % achieved a sufficient mark. In table 1 the antibodies (Abs) used and marks are summarized.

Table 1. Abs and assessment marks for CD20, run 35

1) Proportion of sufficient stains (optimal or good), 2) Proportion of sufficient stains with optimal protocol settings only, see below.

Following central protocol parameters were used to obtain an optimal staining:

Concentrated Abs
mAb clone L26: The protocols giving an optimal result were all based on heat induced epitope retrieval (HIER) using either Cell Conditioning 1 (Ventana) (20/34)* TRS pH 9 (3-in-1, Dako) (13/14), Bond Epitope Retrieval Solution 2 (Leica) (11/14), TRS pH 9 (Dako) (10/13), Tris-EDTA/EGTA pH 9 (8/11), Bond Epitope Retrieval Solution 1 (3/5), Citrate pH 6 (3/4), EDTA/EGTA pH 8 (3/3), Cell Conditioning 2 (1/3) or TRS pH 6.1 (1/1) (3-in-1, Dako). The mAb was diluted in the range of 1:75–1:2,000 depending on the total sensitivity of the protocol employed. Using these protocol settings 96 out of 102 (94 %) laboratories produced a sufficient staining (optimal or good).
* (number of optimal results/number of laboratories using this buffer)

mAb clone 7D1: The protocol giving an optimal result was based on HIER using Bond Epitope Retrieval Solution 1 (1/1). The mAb was diluted 1:200.

rmAb clone EP7: The protocol giving an optimal result was based on HIER using Citrate pH 6 (1/1). The rmAb was diluted 1:100.

Ready-To-Use Abs
mAb clone L26 (760-2531, Ventana): The protocols giving an optimal result were all based on HIER using mild or standard Cell Conditioning 1, an incubation time of 8 to 44 min of the primary Ab at 36°C and UltraView (Ventana, 760-500), OptiView (Ventana 760-700) or IView (Ventana 760-091) as the detection system. Using these protocol settings 36 out of 36 (100 %) laboratories produced a sufficient staining.

mAb clone L26 (IR604, Dako): The protocols giving an optimal result were all based on HIER using TRS pH 9, TRS pH 9 (3-in-1, Dako), TRS pH 6.1 or Bond Epitope Retrieval Solution 1 (Leica), an incubation time of 15 to 25 min in the primary Ab and a 2- or 3-step polymer based system, EnVision (Dako K8000/K8002/K5007) or Bond Polymer Refine (Leica) as the detection system. Using these protocol settings 17 out of 17 (100 %) laboratories produced a sufficient staining.

The most frequent causes of insufficient staining were:

- Too low concentration of the primary Ab.
- Omission of HIER.
- pAb giving cross reactivity.

The prevalent feature of the insufficient results was a generally too weak staining of both the normal and the neoplastic cells supposed to be demonstrated, resulting in a negative or a weak staining of the membranes especially in the neoplastic cells the B-CLL (Fig. 2b). The insufficient results were mainly related to the omission of HIER and/or too low concentration of the primary Ab. The polyclonal antibody RB-9013-P showed an unacceptable cross reactivity resulting in false positive staining in the plasmacytoma and in peripheral nerves (Fig. 3b and Fig. 4b). Tonsil and appendix are 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, nerve cells etc.). HIER is mandatory to obtain an optimal reaction. In concordance with previous observations (Run 21 in 2007) HIER in high pH buffers gives the most efficient retrieval of the CD20 epitopes, but an optimal staining could also be achieved with citrate based buffers for HIER, provided the concentration of the antibody was increased.

This was the 3rd assessment of CD20 in NordiQC, as CD20 also was assessed in run 6, 2002 and run 21, 2007 (table 2). The proportion of sufficient results have once again increased. Even though the number of participants has almost tripled since 2002, the proportion of sufficient results has increased from initially 81% to an encouraging 95%.

Table 2: Proportion of sufficient results for CD20 in the three NordiQC runs performed

Conclusion
The mAb clones L26, 7D1 and the rmAb clone EP7 are all useful for the demonstration of CD20. HIER is mandatory to obtain an optimal result. Concentration of the primary Ab should be carefully calibrated. Tonsil and appendix are appropriate controls: The mantle zone B-cells and the germinal centre B-cells must show a very strong staining reaction. No other cells should stain.