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Recommended CD19 protocols  ■  Recommended CD19 control tissue

Assessment Run 35 2012
 

CD19

The slide to be stained for CD19 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 CD19 staining as optimal included:
  • A strong, predominantly membranous staining reaction of the mantle zone B-cells, the germinal centre B-cells, the interfollicular B-cells and the follicular dendritic cells in the tonsil and the appendix.
  • A weak membranous staining reaction in normal plasma cells in the tonsil and the appendix.
  • A moderate to strong membranous staining reaction in virtually all the neoplastic cells of the DLBCL and the B-CLL.
  • An at least weak to moderate membranous staining reaction of virtually all the neoplastic cells of the Pre-B-ALL.
  • No staining reaction of the plasmacytoma (only residual normal B-cells should be demonstrated).
  • No staining staining of all other cell types.

20 laboratories participated in this assessment. 60 % achieved a sufficient mark. In table 1 the antibodies (Abs) used and marks are summarized.

Table 1. Abs and assessment marks for CD19, run 35

Concentrated Abs

N

Vendor Optimal Good Borderl. Poor Suff.1 Suff. OPS2
mAb clone LE-CD19 11 Biocare
BioSite
Dako
Serotec
5 1 5 0 55 % 75 %
mAb clone BT51E 1 Leica/Novocastra 0 0 1 0 - -
Unknown 2 Unknown 1 0 1 0 - -
Ready-To-Use Abs                
mAb clone LE-CD19, IR656 4 Dako 3 1 0 0 100 % 100 %
mAb clone BT51E, PA0843 1 Leica/Novocastra 1 0 0 0 - -
mAb clone MRQ-36,
119M-17
1 Cell Marque 0 0 0 1 - -
Total 20   10 2 7 1 -  
Proportion     50 % 10 % 35 % 5 % 60 %  

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 LE-CD19: The protocols giving an optimal result were all based on heat induced epitope retrieval (HIER) using either TRS pH 9 (Dako) (2/3)*, TRS pH 9 (3-in-1, Dako) (1/2), Cell Conditioning 1 (1/1) or Bond Epitope Retrieval Solution 2 (Leica) (1/1). The mAb was diluted in the range of 1:25–1:200 depending on the total sensitivity of the protocol employed. Using these protocol settings 6 out of 8 (75 %) laboratories produced a sufficient staining (optimal or good).
* (number of optimal results/number of laboratories using this buffer)

Ready-To-Use (RTU) Abs
mAb clone LE-CD19 (IR656 Dako): The protocols giving an optimal result were all based on HIER using TRS pH 9 (3-in-1, Dako), an incubation time of 20 min of the primary Ab and a 2-step polymer based detection system, EnVision Flex (Dako K8000). Using these protocol settings 4 out of 4 (100 %) laboratories produced a sufficient staining.

mAb clone BT51E (PA0843, Leica/Novocastra): The protocol giving an optimal result was based on HIER using Bond Epitope Retrieval Solution 1 (Leica), an incubation time of 20 min in the primary Ab and a 3-step polymer based system, Bond Polymer Refine (Leica) as the detection system.

The most frequent cause of insufficient stains was:

- Too high concentration of the primary antibody.
- Too low concentration of the primary antibody.

The prevalent feature of the insufficient results was a false positive staining rection of the normal T-cells seen with some LE-CD19 products (Fig. 1). 15 laboratories used the mAb clone LE-CD19. 11 of these were using Dako products (RTU or concentrate) on various platforms (Autostainer, Bond, IntelliPATH and BenchMark ULTRA) and none of these showed a false positive staining of T-cells. The remaining 4 laboratories used the mAb clone LE-CD19 from Serotec, Biocare and BioSite and all showed a false positive T-cell staining. The reason for that is unclear. Common for these three products is a high Ab stock concentration, approximately 10 times higher than that from Dako. This may in part be the explanation for this pattern, which underlines the importance of a carefully calibration of the LE-CD19 clone with special attention to the normal T-cells. Though the number is small, the RTU Abs in general did perform well in this assessment, with Dako LE-CD19 RTU achieving sufficient marks in 4 out of 4 cases (100%). One laboratory used the Leica/Novocastras RTU format based on the mAb clone BT51E and achieved an optimal mark. Among the RTU Abs only clone MRQ-36 from Cell Marque failed (1 case).

Conclusion
The mAb clones LE-CD19 and BT51E are useful for the demonstration of CD19. HIER is mandatory to obtain an optimal result. Concentration of the primary Ab should be carefully calibrated with special attention to normal T-cells to be negative. Tonsil and appendix are both appropriate controls: The mantle zone B-cells, the germinal centre B-cells and the follicular dendritic cells must show a strong staining reaction. Weakly positive normal plasma can be seen. No other cells should stain.

Fig. 1a. Normal tonsil showing an optimal staining for CD19 using the mAb clone LE-CD19 from Dako, diluted 1:50, on the Autostainer platform. HIER was performed using TRS pH 9 (3-in-1) (Dako). A strong and distinct membranous staining reaction is seen in virtually all B-cells. T-cells are negative. Fig. 1b. Normal tonsil showing an insufficient staining for CD19 using the mAb clone LE-CD19 from Serotec, diluted 1:500, on the Autostainer platform. HIER was performed using Citrate pH 6. In addition to a moderate to strong staining reaction in the normal B-cells (albeit weaker than that seen in Fig 1a), the majority of T-cells shows a false positive staining reaction.
Fig. 2a. Lymphatic tissue in the appendix showing an optimal staining for CD19 using the mAb clone BT51E (RTU) on the BOND-III platform. HIER was performed using Bond Epitope Retrieval Solution 1. A strong and very distinct membranous staining is seen in virtually all B-cells, while the T-cells are negative. Fig. 2b. Lymphatic tissue in the appendix showing an insufficient staining for CD19 using the mAb clone BT51E, diluted 1:30, on the BenchMark platform. HIER was performed using Cell Conditioning 1. Only a weak to moderate staining is seen in the majority of B-cells. T-cells are negative. Also compare with Fig. 3b, same protocol.
Fig. 3a. Optimal staining reaction for CD19 of the DLBCL. Same protocol used as in Fig. 2a based on the mAb clone BT51E. A moderate to strong membranous staining reaction is seen in virtually all the neoplastic cells. Fig. 3b. Insufficient staining reaction for CD19 of the DLBCL using same protocol as in Fig. 2b. Only a weak staining is seen in scattered neoplastic cells. The majority of the tumour cells are negative. Compare with the optimal protocol in Fig. 3a, same field.
ON/SN/MV/LE

Last update 13-07-2012