It is important to determine the relative and absolute numbers of the various lymphocyte subsets to ascertain if a sample is abnormal. The lymphocyte population consists of three major types: T lymphocytes (CD3+), B lymphocytes (CD19+) and Natural Killer lymphocytes (CD56+/CD3–); each of these groups can be further subdivided, e.g., CD4+ T lymphocytes, CD8+ T lymphocytes, CD5+/– B lymphocytes, CD8+/– NK lymphocytes. A 9 color panel of 10 antibodies was created, which allowed easy identification of each lymphocyte group and some of their sub types.
Currently for lymphocyte subset enumeration, it is typical to use a Lyse No Wash technique, in which cells are stained with antibody for 10 –15 minutes, after which red blood cells are removed by adding a lysing reagent. After approximately 10 –15 minutes incubation, the sample is acquired on the flow cytometer without prior washing. We have developed a technique in which samples can be acquired using a CyAn ADP 9 Color flow cytometer, without lysing the red blood cells, saving 10 –15 minutes in preparation time. Sample acquisition time on the instrument can also be reduced significantly by using high sample flow rates of 150 –200 μL/min.
A gating strategy has been developed that permits the removal of the majority of non-lymphocyte events, allowing easier identi fication of the lymphocyte populations and their subsets. The addition of reference counting beads also allows for the single platform determination of absolute cell counts.