The 52nd EBMT Annual Meeting was held in Madrid, Spain, from March 22 to 25, 2026. The conference focused on the latest advances in clinical hematology and hematopoietic stem cell transplantation, promoting continued progress in both clinical practice and translational research. In this issue, Prof. Hui Wang presents recent advances in flow cytometric immunophenotyping for acute promyelocytic leukemia (APL), based on the study entitled “An early myeloid stem cells-based immunophenotypic panel can rapidly and efficiently distinguish APL from APL-like.”
Background
Typical cases of acute promyelocytic leukemia (APL) exhibit characteristic morphology and immunophenotype. However, approximately 10%–20% of cases present with atypical features. In addition, certain non-APL acute myeloid leukemia (AML) cases—particularly those with NPM1 mutations or KMT2A rearrangements—can display morphology and immunophenotypes resembling APL; these are referred to as “APL-like.”
Distinguishing APL from APL-like AML therefore represents a major diagnostic challenge in flow cytometry. To address this, we developed a two-tube, eight-color panel based on early myeloid stem/progenitor cell immunophenotypes and constructed a quadrant-based analytical map to enable rapid and efficient differentiation between APL and phenotypically similar non-APL cases.
Methods
A total of 313 patients with APL diagnosed at Yanda Lu Daopei Hospital between January 1, 2016, and December 20, 2024, were included (165 males and 148 females). Among these, 236 had classical APL (124 males, 112 females), and 77 had variant APL (APLv; 41 males, 36 females).
The APL-like cohort consisted of 426 patients, including 311 with NPM1-mutated AML and 115 with AML harboring other genetic abnormalities (219 males and 207 females).
All cases were validated using integrated morphological, genetic, and clinical data.
A two-tube, eight-color flow cytometry panel was used:
- Tube 1: CD16 FITC / CD117 PE / CD34 PerCP-Cy5.5 / CD13 PE-Cy7 / CD33 APC / HLA-DR APC-Cy7 / CD11b BV421 / CD45 V500
- Tube 2: MPO FITC / CD64 PE / CD34 PerCP-Cy5.5 / CD117 PE-Cy7 / CD14 APC / CD36 APC-Cy7 / CD38 BV421 / CD45 V500
Early myeloid stem/progenitor cells (EMSCs) were defined as: CD34⁺ / SSC^dim / CD117^dim / CD33^dim / HLA-DR^dim / CD13⁺ / CD11b⁻ / CD38^dim
Results
1. Detection of Early Myeloid Stem/Progenitor Cells (EMSCs)
EMSCs were detected in only 2 of 313 APL cases (0.64%), including 1 of 236 classical APL cases (0.42%) and 1 of 77 APLv cases (1.30%). In contrast, EMSCs were detected in 423 of 426 APL-like cases (99.30%).
Using the presence or absence of EMSCs to distinguish APL from APL-like AML yielded a sensitivity of 99.36% and a specificity of 99.30%. The positive predictive value of “absence of EMSCs” for diagnosing APL was 99.04%, and the negative predictive value was 99.53%.
When combined with conventional markers such as MPO, CD34, and HLA-DR, this approach enables rapid, efficient, and reliable differentiation between APL and APL-like AML.
2. Additional Immunophenotypic Features
The APL group showed relatively consistent immunophenotypic patterns. Typical APL cases expressed CD64 and MPO, while lacking expression of HLA-DR and CD34.
Interestingly, CD34 expression was highest in APLv cases, exceeding that observed in APL-like AML. HLA-DR remained a highly informative marker for APL, with a very low positivity rate. However, many APL-like cases exhibited immunophenotypes highly similar to APL.
Due to the heterogeneity of APL-like AML, markers such as CD64, MPO, CD34, and HLA-DR showed statistically significant variability.
3. Quadrant-Based Analytical Model
A quadrant-based diagnostic map was constructed by integrating EMSC detection with immunophenotypic markers, using MPO as a reference axis.
Two marker groups were defined: MPO/HLA-DR and CD34/SSC.
- South (MPO⁻ HLA-DR⁺): SSC and CD34 positive or negative → non-APL
- East (MPO⁺ HLA-DR⁺): low SSC, CD34 positive or negative → non-APL
- West (MPO⁺ HLA-DR⁻): low SSC, CD34⁻ → APL-like
- North (MPO⁺ HLA-DR⁻): high SSC, CD34⁻ → typical APL
- Northwest (MPO⁺ HLA-DR⁻, low SSC, CD34⁺): APLv
- Northeast (MPO⁺ HLA-DR⁺, high SSC, CD34⁻): rare HLA-DR⁺ APL
Most cases in the “north” region lacked EMSCs, whereas most cases in the “south” region showed EMSC presence.
This framework may serve as a prototype for future artificial intelligence–assisted diagnostic models.
Conclusion
This study establishes a multiparametric immunophenotypic approach based on early myeloid stem/progenitor cell detection. When combined with a quadrant-based analytical model, it enables rapid and accurate differentiation between APL and phenotypically similar leukemias.
This approach also has potential as a foundation for AI-assisted diagnostic systems.
Investigator Profile
Hui Wang, PhD Lu Daopei Hospital
Prof. Wang is a senior researcher and serves as Deputy Director (vice president level) of the Beijing Lu Daopei Institute of Hematology, as well as Deputy Director of the Department of Laboratory Medicine at Lu Daopei Hospital.
She is an editorial board member of the Chinese Journal of Laboratory Medicine and a young editorial board member of the Chinese Journal of Hematology. She holds leadership roles in multiple national academic organizations, particularly in flow cytometry and laboratory diagnostics.
With 25 years of experience in flow cytometry, she has independently issued over one million clinical reports. She holds 11 Chinese invention patents and 3 U.S. patents. She has led the development of 7 expert consensus statements and contributed to 12 additional guidelines and standards.
She has published more than 60 SCI-indexed and core journal articles as first or corresponding author and has contributed to over 100 publications overall.
