Flow cytometric clonal excess analysis of peripheral blood, routine handling, and pitfalls in interpretation

Clonal excess (CE) analysis by flow cytometry is a convenient method to detect minimal involvement of peripheral blood and bone marrow by B-cell lymphoma. The method is based on evaluation of the congruity between kappa and lambda light chain distributions within a normal B-cell population. By using the Kolmogorov-Smirnov method for evaluation of histogram identity, the maximum difference (D value) between distributions is calculated. However, variable adsorption of cytophilic plasma immunoglobulin (Ig) to CD16 positive cells, T-cell subsets, and the B cells themselves may cause incongruity between the light chain distributions that might simulate or disguise a true clonal excess and thus create major pitfalls in the interpretation of the CE analysis. These phenomena are observed both in normal blood donors and in patients with non-Hodgkin's lymphoma. In the present report we describe: (1) how freezing of the isolated mononuclear cells before immunostaining effectively removes most of the adsorbed cytophilic Ig, (2) how by exclusion of the CD16 positive population further resolution of the relevant histogram shape is obtained to avoid false interpretations of incongruity, and (3) how adsorption of Ig to B-cells creates a typical pattern of "reciprocal labeling" that can be mistaken for a clonal excess. Based on our observations we argue against an uncritical use of normal D values for definition of clonal excess and advocate an analytic evaluation of the kappa and lambda overlay distribution by dual immunofluorescence, either by excluding CD16 positive cells or by including only B cells, to reveal the nature of the different deviations.