Stage 0 hematogones essentially lack CD9 expression: comparative immunophenotypic analysis of hematogone subsets and myeloid blasts

Measurable residual disease (MRD) assessment of B lymphoblastic leukemia (BLL) by flow cytometry (FC) can be confounded by normal populations, particularly stage 0 hematogones (HGs), following CD19-targeted therapy. Few studies have reported expression patterns or compared them with other populations. We assessed expression of CD9, CD34, CD45, CD58, CD66c/CD13/CD33, and CD81 on stage 0 HGs and compared the findings to stage 1 to 3 HGs and myeloid blasts (MBs).

Cases of BLL MRD FC were reviewed for residual disease, and negative cases with stage 0 HGs were further evaluated for stage 1 to 3 HGs and MBs. Dot plot patterns and median fluorescence intensity (MFI) were evaluated and compared across populations.

Unlike stage 1 to 3 HGs, stage 0 HGs essentially lacked CD9 expression. CD9 MFI on stage 1 to 3 HGs demonstrated a broad range, typically with prominent bright components, although some cases showed significant CD9-negative subsets. Stage 0 HG CD34 was similar to stage 1 and slightly lower than MBs. CD45 expression was similar among stage 0 HGs, stage 1 HGs, and MBs. Although all HG stages appeared overall similar on dot plots, CD58 and CD66c/CD13/CD33 MFIs were higher, and CD81 MFI was lower in stage 0 HGs than in later stages. CD81 expression was higher in all HG stages compared with MBs.

Unlike stage 1 to 3 HGs, stage 0 HGs essentially lack CD9 expression. CD58, CD66c/CD13/CD33, and CD81 may show slight shifts in intensity compared with other stages. These findings should be considered when assessing BLL MRD by FC following CD19-directed therapy.