The optimal flow cytometry analysis approach for biologic studies aimed at investigating the earliest stages of leukemogenesis will depend on the numbers of abnormal cells required for validation and the planned downstream analysis. Studies investigating CLL-like MBL designed to detect at least 1 CLL cell/μL will require analysis of at least 200,000 total events using a minimum of five color analysis (CD5, CD19, CD20, kappa, lambda). In many cases the detection of an abnormal population can be confirmed by consensus IGH-PCR on separated B-cells. Analysis of CLL cells below this level will typically require analysis of more than 500,000 total cells with six or more fluorescent channels (CD3, CD5, CD19, CD20, kappa, lamda). Such studies need to be carefully controlled and validation of the abnormal population requires fluorescent cell sorting of the abnormal population coupled with FISH analysis and IGHV gene sequencing after cloning or extraction of DNA from single sorted cells.(15)
