Evaluation of Flow Cytometric Methods Used in Analysis of Immune Cells in Patients with Malignant Lymphoma.

Abstract: Malignant lymphomas are a group of cancerous diseases that develop from lymphocytes and primarily affect lymph nodes. Being the sixth most common cancer type in Sweden, lymphoma is a societal problem that needs to be tackled by improving care and treatment of patients. This study was designed to examine the blood cell composition in lymphoma patients and well as determine whether the use of cryopreserved cells affected the analysis outcome. An evaluation of the methods used was also performed. Frozen peripheral blood from lymphoma patients as well as fresh and frozen blood from healthy controls was used. The cells of interest were monocytes, granulocytes, Treg, NKT, iNKT, B and T cells plus the dendritic cell activation protein CCR7. Three immunophenotyping methods were used. Method one was used in staining surface cell markers while the other two were for both surface and intracellular staining using two distinctive kits. The results showed no significant difference in immune cell composition between patients and blood donors. Limited patient samples and the lack of female blood donors could explain the unexpected result. A substantial difference in Treg cells was observed in fresh and frozen tested samples as well as T cell outcomes in method one compared to the other two methods. There were fewer Treg cells in frozen samples, which probably was due to cryopreservation while the lack of fixation in method one led to the loss of CD4+ T cells. Overall, the methods used were adequate but definitely require some improvements.