Authors' Response to Letter from Khanolkar and Ahmed concerning our article “An unusual pattern observed upon the addition of <scp>CD79b</scp> to a flow cytometry B‐cell lymphoma panel” (Panakkal et al., 2025). <i>Cytometry Part B: Clinical Cytometry

The authors appreciate the thoughtful comments by Drs. Aaruni Khanolkar and Aisha Ahmed and agree that the pattern described in our Letter to the Editor “An unusual pattern observed upon the addition of CD79b to a flow cytometry B-cell lymphoma panel” (Panakkal et al., 2025) is not very uncommon. However, it may be unexpected in flow cytometry laboratories with limited experience in evaluating CD79b expression and represents a challenge to flow cytometry laboratories in terms of differentiating such physiological patterns from artefactual causes of cells appearing on the so-called “line of death.” Drs. Khanolkar and Ahmed are correct. We did not include the clone of the CD79b antibody we used for the short format of a Letter to the Editor. They correctly inferred that we used clone 3A2-2E7 (also known as SN8) from BD Biosciences. We have not evaluated other clones and therefore cannot comment on any potential clone-related differences in the pattern observed. While tandem assessment of CD20 and FMC7 may also lead to cells appearing on the diagonal, this situation represents the use of two antibodies that target the same molecule. FMC7 is a clone known to bind to CD20 (Deans & Polyak, 2008). We are unaware of how common CD24hiCD38hi CD19+ B cells appear along the diagonal. The CD19/CD21 pattern is not one we have observed in our laboratory. Our “memory B cell” panel has CD19 BV786 and CD21 PE-Cy7, which when plotted together, the B cells do not appear along the diagonal (Figure 1a). Some “angulation” of clusters is seen relatively frequently in daily practice. Even plotting CD19 versus CD20 on normal B cells can show an angular pattern (Figure 1b). The basis for the diagonal “tail” in such displays could be that the expression of both markers in the subset displayed is particularly bright. We also have not observed a diagonal pattern when CD20 and anti-Ig light chain antibodies are combined in the same tube, and we suspect that we are not alone in this regard. Most clinical laboratories that perform evaluation of hematolymphoid neoplasms by flow cytometry use the combination of CD20 and anti-Ig light chain antibodies in the same tube. The rapid dissociation of B-cell receptor from CD20 upon cross-linking could be why the combined use of CD20 and anti-Ig light chain does not cause a diagonal staining pattern (Polyak et al., 2008). While the use of CD79a may resolve the appearance of the diagonal pattern, it requires fixation and permeabilization, which adds time and complexity to the assay. Most importantly, it does not directly address the interest pertaining to CD79b expression on the surface of the cells for patients who are receiving or may receive polatuzumab vedotin that targets CD79b. Finally, we thank Drs. Khanolkar and Ahmed for sharing their experience. We believe this is valuable and, along with providing plausible explanations for such findings, can help other laboratories during assay development, validation, and troubleshooting and broaden the understanding of the biology behind the assays in use in clinical flow cytometry laboratories.