Monoclonal Igκ and Igλ Antibodies Minimize Cross-Reactivity With NK Cells In a Multicolor Immunophenotyping Panel

Immunophenotyping provides a valuable aid to leukemia and lymphoma diagnosis and classification. Comprehensive initial screening panels must include markers that identify all major cell populations, such as T cells, B cells, and NK cells. It also is useful to further phenotype the subsets of the major populations within the same tube. This can be achieved by using a single fluorochrome for two markers that are specific for subsets of different cell types (for example, CD8 FITC for NK cells and Igλ FITC for B cells). Such a cocktail minimizes the amount of patient sample required for classification while simultaneously decreasing workflow, reagent consumption, and time.

The current study examines the interaction between Igλ FITC, Igκ PE, CD8 FITC, and CD56 PE surface staining. We identified that Igλ and Igκ polyclonal antibodies cause an artificial dragup of the CD8 FITC NK population in a fraction of healthy donors. This dragup results in the majority of NK cells appearing to be CD8⁺, which is consistent with the phenotype of chronic lymphoproliferative disorders of NK cells [Am J Clin Pathol 127:881; Br J Haematol 120:1026].

Since this artificial dragup may indicate an inappropriate result for the patient specimen, we assessed the effects of different Igκ and Igλ antibodies on this stain. The highly conserved nature of Igλ and Igκ across species caused us to evaluate polyclonal antibodies column-adsorbed against cross-reactive antibodies in addition to total polyclonal and monoclonal antibodies. We conclude that monoclonal Igκ and Igλ antibodies minimize the phenotypic effects on the NK population when using the same fluorochromes, while providing sufficient separation to discriminate Igκ⁺ and Igλ⁺ B cells. These studies decrease the risk of errors in disease classification in assays that simultaneously evaluate subsets of multiple cell types.