Multiplexing 8 colors with 12 antibodies in a single lymphoid screening tube by flow cytometry for evaluating suspected chronic lymphoproliferative disorders (CLPD)

The diagnosis of chronic lymphoproliferative disorder (CLPD) or non-Hodgkin Lymphoma (NHL) is based on the detection of the abnormal clonal lymphoid cells. The flow cytometry (FCM) immunophenotyping not only plays an essential role in the screening of CLPD but also helps in the specific identification and characterization of the expanded aberrant lymphocytes. Over decades, it has evolved from a single parameter to multi-parameter assessment by 3- to 12-color FCM. The greatest challenge is to characterize abnormal lymphoid cells by a limited immunophenotype (IPT) panel. A study was undertaken to evaluate the diagnostic usefulness of a single lymphoid screening tube (LST) FCM assays that included a multiplex of 12 antibody cocktails consisting of CD45, CD19, CD3, CD4, CD8, TCRγδ, CD5, CD20, CD56, CD38, kappa, and lambda in an 8-color labeling based on the proposed EuroFlow (EF) antibody panels with slight modifications. We have included the same set of markers and fluorochrome combinations except for CD45 V500-C, CD4 V450, and CD20 V450 instead of CD45 Pacific Orange (PacO), CD20 Pacific Blue (PacB), and CD4 PacB. A logical gating strategy was used to separate different subsets of lymphoid populations for the detection of the clonal lymphoid cells. Further, antibody cocktails were used depending upon the nature of the clonal population like B, T, or natural killer (NK) cells for definitive classification. The results were further correlated with trephine biopsy, immunohistochemistry (IHC), and molecular or cytogenetics wherever required for definitive characterization. A total of 82 consecutive samples with suspicion of CLPD or NHL were analyzed by a 3-laser 8-color flow cytometry. Out of which 73 cases were diagnosed to be CLPD, 2 cases were diagnosed as acute lymphoblastic leukemia (ALL), and 7 cases had polyclonal lymphoid population. We could achieve a diagnostic sensitivity of 100% in the diagnosis of CLPD. In addition, the average number of tubes as well as antibodies was reduced by one-third in comparison to a standard protocol. The development of 8-color and 12-antibody LST by flow cytometry can provide maximum IPT information and meet economical requirements especially in developing countries by reducing the total cost of antibodies, labor, and time.