Scientific Calendar September 2023

Morphological analysis using a DI-60 automated digital imaging analyser revealed the presence of smudge cells.

The patient's blood analysis revealed a markedly increased WBC count of 26.17 x10³/µL, of which 80% were lymphocytes (LYMPH# 21.15 x10³/µL). This is visible in the WDF scattergram where the lymphocyte population presents as a very dense cloud.

The reagents used in the WPC channel have a stronger lysis effect on the membranes of the WBC than the ones used in the WDF channel, this leads to a higher cell permeability. The specific fluorescent marker used in the WPC channel labels the DNA inside the cell’s nucleus. The higher the degree of permeabilisation, the more fluorescent marker can enter the cell and bind to the DNA, causing a high fluorescence signal. Neoplastic lymphocytes, which are more mature, have readily permeated membranes, due to the higher lipid content of the cell membranes, and therefore give off a high fluorescence signal. This can be seen in the WPC scattergram in Figure 1: the population of neoplastic lymphocytes form a cluster of red dots in the high fluorescent area of the WPC scattergram. The SSC-FSC view of the WPC scattergram shows two distinct lymphocyte populations (blue circles).

The sample was pre-washed before staining, to exclude extraneous immunoglobulin in the plasma, which could affect the final results. After this, a lyse/wash protocol was performed. In short:

• A cocktail of antibodies was placed into a 75 mm x 12 mm polystyrene tube
• 100 µL of the washed sample was aliquoted into the same tube and vortex mixed at 2,500 rpm for 5 seconds
• Incubation for 15 minutes in the dark at room temperature
• 2 mLs of 1 in 10 diluted CyLyse FX was placed into the tube, to lyse the red cells and fix the white cells, vortex mixed at 2,500 rpm for 5 seconds
• Incubation for 10 minutes in the dark at room temperature
• The sample was then washed twice with PBS and placed on the XF-1600 for acquisition

Antibody/antigen interactions take place in the sample and cocktail mixture so that the monoclonal antibodies attach to the specific target antigen on the cell surface. Each monoclonal antibody has a fluorescent label tag, which the flow cytometer detects, allowing the user to distinguish what is positive and negative, therefore allowing for a diagnosis to be made.

The CyLyse FX is a formaldehyde-based lyse solution which lyses red cells and fixes the white cells with the antibody and tag attached. Without the lyse, the flow cytometer would also acquire red cells, which are not relevant for this test.

Finally, the sample is washed to exclude the red cell debris from being acquired, this allows the user to have a cleaner picture during acquisition of the sample and ensures accurate data analysis.

In this case, the immunophenotyping revealed a lymphocytosis and a dim CD19/CD5 dual-positive population. In addition, a monoclonal proliferation of the lambda light chain was observed. The sample also expressed CD20, CD23 and CD79b and was negative for CD10, CD11c and CD38, all of which point to the diagnosis of a B-CLL.