Measurable Residual Disease Assessment in B-Lymphoblastic Leukemia Using 5- and 10-Color Flow Cytometry: An...

Measurable residual disease (MRD) assessment by flow cytometry is an established prognostic tool in B-lymphoblastic leukemia (B-ALL). Advances in multicolor flow cytometry have enabled higher event acquisition and expanded immunophenotypic analysis; however, real-world data comparing 5-color and 10-color flow cytometry platforms remain limited.

To evaluate MRD detection by 5-color and 10-color flow cytometry and to assess leukemia-associated immunophenotype (LAIP) expression patterns and concordance with post-induction morphology in patients with B-ALL.

This retrospective study included patients with B‑ALL who had diagnostic immunophenotyping and post‑induction MRD assessment between 2018 and 2024 at a single center. MRD was evaluated using predefined LAIPs on a 5‑color, lower‑event acquisition protocol and a 10‑color, higher‑event acquisition protocol. MRD was considered positive when the presence of ≥10 clustered aberrant events was identified, showing a coherent population with two or more abnormal antigen expression patterns. Clinical, morphological, and flow cytometric parameters were collected and analyzed.

A total of 172 paired baseline and post‑induction MRD samples were studied. MRD was detected in 38 cases (22.1%), with similar overall positivity rates on the 5‑color (12, 23.5%) and 10‑color (26, 21.5%) platforms. CD58 overexpression emerged as the most reproducible LAIP on both assays, while the 10‑color panel allowed finer assessment of treatment‑related antigen shifts, including loss of CD10 and CD34 in a subset of MRD‑positive samples, and achieved lower limits of MRD detection. Notably, more than two‑thirds of MRD‑positive cases by flow cytometry were classified as negative on conventional morphology.

In this cohort, 5‑color flow cytometry provided practical MRD assessment in B‑ALL, whereas 10‑color flow cytometry offered greater immunophenotypic detail and improved recognition of low‑level residual disease. These findings support the routine use of flow cytometry‑based MRD evaluation alongside morphology for post‑induction response assessment and risk‑adapted management in B‑ALL.