Utility of flow cytometry for immunophenotyping double-hit lymphomas


We read with interest the publication by Platt et al. who recently considered the flow cytometry features of double-hit B-cell lymphomas in a four-color platform [1]. This report was a follow-up to our study that proposed a characteristic immunophenotype of so-called, “double-hit” B-cell lymphomas [2] that was independently substantiated Harrington et al. [3].

We appreciate the detailed work of Platt et al. but wonder if they did not find similar features of double-hit lymphomas in their four-color platform due to differences in approaches. Platt et al. report using, “resting, comingling polytypic B-cells,” as compared to our (and Harrington et al.'s) use of germinal center B cells as a reference population. In our study, germinal center B cells were chosen as the reference population as double-hit lymphomas have a germinal center immunophenotype with expression of CD10 and BCL-6 [4, 5]. Because germinal center B cells express CD20 at a higher level than other B cells, this methodologic difference may have resulted in lower sensitivity in the work by Platt et al. to identify this feature. Further, in our study, we used an eight-color, single-tube multiparametric flow cytometry assay. We were able therefore concurrently to evaluate the immunophenotypic expression of CD5, CD10, CD45, and CD38 in addition to CD19, CD20, and surface immunoglobulin light chains. Prior studies have firmly established that C-MYC positive tumors have high expression of CD38 [6, 7]. Expression of CD38 was not apparently considered by Platt et al. and may have limited their ability to identify double-hit lymphomas. Lastly, Platt et al. conclude that dim CD20 is not a consistent feature of “double-hit” lymphomas; however, their use of the comparatively dimmer fluorochrome-Per-CP (vs. V450 used in our study) may also have limited the ability to detect subtle differences in CD20 expression.

In short, we suggest that these methodologic differences may have resulted in the conclusion by Platt et al. that flow cytometry is of limited utility to detect double-hit lymphomas. Of note, Harrington et al.'s work, whose methodology more closely resembled ours, supported our findings. Importantly, the work of Platt et al. implies that laboratories using a similar approach (absence of evaluation of CD38, resting B cells as reference population, and dimmer CD20 fluorochromes) may also not reliably identify double-hit lymphomas.

  • David Wu

  • Brent L. Wood

  • Russell Dorer

  • Jonathan R. Fromm

  • Department of Laboratory Medicine, University of Washington Medical Center, Seattle, WA

  • Department of Pathology, Virginia Mason Medical Center, Seattle, WA