1. When using the StrABC/HRP technique for each primary antibody, the pretreat-ment protocol and dilution required need to be established for each user, so that the best possible demonstration of antigen is achieved with minimal nonspecific binding, which is assessed using a negative control and by examination of internal cellular controls within the positive control and the test. Positive controls should be used for each antibody.

2. On paraffin sections, CD10 and Bcl-6 work optimally using the standard StrABC technique with the DAKO ChemMate Kit to detect the antigen; however, tyramide signal amplification can be used to intensify the staining in resin-embedded samples or if weak staining is observed by routine methods.

3. With the TSA technique, the Dako CD23 clone (MHM6) requires tyramide signal amplification to optimize staining; however, the clone available from Novocastra and Serotec (1B12) produce optimal staining using the standard StrABC technique.

4. The reliability of immunocytochemistry methods is critically dependent on the initial handing of the tissue specimen. It is important that lymph node biopsies are sent to the laboratory unfixed and as quickly as possible.

5. Placing a whole specimen in formalin results in major artifacts, with wide variation in fixation between the center and edge of the node. As a result, the morphology and intensity of tinctorial and immunocytochemical staining are highly inconsistent across the tissue section. This is particularly apparent with many of the antibodies used for demonstrating a germinal center phenotype, and in extreme cases the diagnosis may be compromised if the specimen is poorly fixed. To avoid this, tissue should be thinly sliced into pieces of no more than 2- to 3-mm thick.

6. There has been a great deal written on methods of fixation of lymphoid tissue for the optimization of immunocytochemical staining. When modern immunocy-tochemistry is used, 10% formalin or neutral buffered formalin is adequate for all routine applications, and "special" fixatives are no longer required.

7. The advantage of pressure-cooking is that multiple 25-slide racks can be pressure-cooked at the same time and, therefore, it is less time-consuming than microwaving. Pressure-cooking also can result in superior demonstration of certain antigens. Pressure-cooking can, however, lead to poor morphology and inaccurate staining in suboptimally fixed specimens, particularly in very small biopsies in which fixation times are reduced. Microwaving is recommended in these situations.

8. If the Dako ChemMate kit is to be used, prepare ChemMate DAB solution by adding 20 |L of DAB concentrate (reagent C) per 1 mL of substrate (reagent D), allowing 200 |L of DAB solution per slide. Alternatively, use DAB working solution as described in the immunocytochemistry materials section.

9. When performing flow cytometry lysis also may be performed at room temperature, but this may be suboptimal if samples are older than 24 h. Complete lysis may take longer than 5 min, but leucocyte degradation begins to occur after 20 min.

10. For Becton Dickinson flow cytometers, initial calibration is best performed using FACSComp with CALIBrite beads, and then further calibrated using relevant reagents from the panel on cells. In particular, for CD19 PE/Cy5 reagents, an aliquot of cells should be stained with this reagent alone, and compensation optimized accordingly.

11. This number of cells will be adequate for immunophenotyping B-lymphocyte populations that represent more than 1% of leucocytes. For smaller populations, more accurate results may be obtained if 50,000 events are acquired. However, more specific antibody combinations may be required to detect minimal disease.

12. T-cell contamination: these cells are rarely a problem, and frequent contamination of a B-cell region by T-cells is usually indicative of a poor CD19 reagent. However, the expression of CD19 may be weak in follicular lymphoma/DLBCL, and in some very rare cases it may be necessary to repeat the analysis using a different B-cell gating reagent such as CD20. T-cell contamination is identifiable as a population of cells that bind both CD3 FITC and CD3 PE in equal proportions. In tissue biopsies, T-cells are often present in the B-cell gate at low level because of cell:cell adhesion of B-cells to T-cells that occurs in follicles.: these cannot be excluded by this gating strategy, but it is extremely rare that these represent more than 1% of events in the B-cell gate.

13. Apoptotic cell contamination: these are problematic because they show variable levels of nonspecific binding. Again, fluorescein isothiocyanate conjugates are most likely to bind nonspecifically. However, antibodies conjugated to other fluo-rochromes may also show very high levels of nonspecific binding to apoptotic cells, and there are also differences between identical antibodies conjugated by different companies. It is therefore necessary to exclude all apoptotic cells, including apoptotic B-cells, because their inclusion may lead to a false-positive result for certain antigens. Apoptotic cells frequently are present in bone marrow samples, particularly those in which normal B-cells also are present. In some tissue biopsies, virtually all of the B-cells may be apoptotic or necrotic, but this can be minimized by gentle disaggregation techniques and by analysis immediately after disaggregation. For definitive exclusion of apoptotic cells, a viability dye such as propidium iodide or preferably 7-AAD may be included, but this will limit the number of channels available to analyze antigen expression. In the panel used here, binding of CD3 FITC but not CD3 PE is indicative of contamination with apoptotic cells. In most case, they may be completely removed by adjusting the scatter region to gradually remove cells with the lowest forward scatter until there is no evidence of aberrant binding.

14. Monocyte contamination: these cells have high levels of nonspecific binding and as such may appear to have weak CD19 binding. The are readily separated from most B-LPDs by their high side scatter but often show overlapping characteristics with DLBCL cells. They also are present in bone marrow/peripheral blood samples or tissue samples with heavy blood contamination. Immunoglobulin is present on the surface of monocytes ligated to Fc receptors. Both normal and neoplastic B-cells express only one light chain or none at all, whereas mono-cytes will have both kappa and lambda bound to their cell surface. As such, assessment of kappa vs lambda binding will reveal a diagonal line if monocytes are present, with B-cells to the upper left, lower right, or lower left of the plot. In addition, monocytes tend to bind FITC antibodies nonspecifically to a greater extent than PE antibodies. Therefore, a population of cells with moderate side scatter, weak binding of several FITC reagents, and equivalent binding of both kappa and lambda antibodies should be excluded from analysis. This can be achieved by adjustment of gating regions allowing exclusion of monocytes without removal of any B-cells in the vast majority of cases.


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