Editor's Note: Differential diagnosis of chronic lymphoproliferative disorders (CLPD) remains a major challenge in hematology. Given the blurred boundary between reactive lymphoid proliferation and clonal proliferation, as well as the substantial overlap in morphological and immunophenotypic features among different subtypes, avoiding misdiagnosis and missed diagnosis is of critical importance. During the 2026 Cross-Strait Hematology Academic Conference, Oncology Frontier – Hematology Frontier invited Professor Wei Xu of the First Affiliated Hospital of Nanjing Medical University (Jiangsu Province Hospital) to systematically explain the diagnostic approach integrating basic morphology, flow cytometric immunophenotyping, and molecular genetic testing. Professor Xu also provided an in-depth analysis of strategies for resolving challenging CLPD cases, offering authoritative guidance for clinicians to establish a standardized, multidimensional, and precise classification system for these disorders.《Oncology Frontier – Hematology Frontier》: Reactive lymphocytosis and clonal lymphoproliferative disorders can easily be confused in clinical practice and represent a major challenge during initial diagnosis. Could you discuss the core principles for distinguishing the two? What clinical manifestations and routine laboratory findings can help physicians rapidly differentiate benign reactive proliferation from malignant clonal disease and avoid overdiagnosis or missed diagnosis?
Professor Wei Xu: In clinical practice, patients are often referred to the hematology department after an elevated absolute lymphocyte count is detected on a routine complete blood count performed during a health examination or evaluation for another condition. At this point, the primary task for the hematologist is to determine whether the lymphocytosis is reactive or clonal.
For this differential diagnosis, the most basic and efficient clinical strategy is to perform peripheral blood smear morphology and flow cytometric immunophenotyping simultaneously. In flow cytometric analysis, for B-cell disorders, clonality is primarily assessed by determining whether there is restricted expression of either the κ or λ light chain. For T-cell disorders, attention should be paid to pan-T-cell antigens such as CD2, CD3, CD5, and CD7. Loss or weak expression of commonly expressed antigens such as CD7 or CD5 may suggest the presence of an abnormal T-cell clone.
Molecular testing is also an important diagnostic tool. At the B-cell level, immunoglobulin heavy-chain (IGH) or light-chain (IGκ, IGλ) gene rearrangement can be assessed to evaluate clonality. At the T-cell level, T-cell receptor (TCR) gene rearrangement testing can be used to determine whether clonal proliferation is present.
In summary, for patients with lymphocytosis, peripheral blood smear examination, flow cytometric immunophenotyping, and molecular testing can be integrated to distinguish reactive lymphocytosis from clonal lymphocytosis.
《Oncology Frontier – Hematology Frontier》: B-cell chronic lymphoproliferative disorders have substantial overlap among subtypes. Diseases such as chronic lymphocytic leukemia, mantle cell lymphoma, hairy cell leukemia, and Waldenström macroglobulinemia can present with highly similar morphological and clinical features, making them particularly challenging to differentiate. Could you summarize the key criteria for precise diagnosis and the most commonly confused disease pairs and their distinguishing features?
Professor Wei Xu: B-cell chronic lymphoproliferative disorders (B-CLPDs) comprise a highly heterogeneous group of diseases, mainly including chronic lymphocytic leukemia (CLL), follicular lymphoma (FL), marginal zone lymphoma (particularly splenic marginal zone lymphoma, SMZL), mantle cell lymphoma (MCL), lymphoplasmacytic lymphoma/Waldenström macroglobulinemia (LPL/WM), and hairy cell leukemia (HCL). These disorders share the common feature of increased peripheral blood lymphocyte counts, with clonal B-cell proliferation confirmed by laboratory testing. Further differential diagnosis is therefore required.
Flow cytometric immunophenotyping is commonly used for differential diagnosis, as different subtypes exhibit characteristic immunophenotypes. For CLL, for example, the Royal Marsden Hospital (RMH) flow cytometric scoring system is commonly used, incorporating markers such as CD5, CD23, CD22, CD79b, FMC7, and surface immunoglobulin (sIg). A score of 4–5 supports a diagnosis of CLL. Although MCL also expresses CD5, CD20 expression is typically stronger, while CD23 is usually negative or weakly expressed. For LPL/WM, flow cytometry can identify a clonal plasma-cell population, while FL originates from germinal-center B cells and can be differentiated using germinal-center-associated markers such as CD10. Through immunophenotypic analysis, the majority of B-CLPD cases can be accurately classified.
For challenging cases that cannot be definitively diagnosed by flow cytometry, bone marrow immunohistochemistry (IHC) and molecular genetic testing should be incorporated into a comprehensive assessment. On immunohistochemistry, LEF1 is a highly specific marker for CLL and is positive in the vast majority of CLL cases; a negative result can therefore help exclude the diagnosis. For MCL, Cyclin D1 and SOX11 positivity have important differential diagnostic value. At the molecular genetic level, MCL is characterized by the t(11;14) chromosomal translocation, which can be detected by fluorescence in situ hybridization (FISH) to support the diagnosis. More than 90% of patients with LPL/WM harbor the MYD88 L265P mutation, making this molecular marker highly valuable for diagnosis.
HCL can likewise be diagnosed using multiple complementary approaches. Flow cytometry typically demonstrates expression of CD103, CD123, CD11c, and CD25. Morphologically, characteristic cells with hair-like cytoplasmic projections can be observed in peripheral blood or bone marrow smears. At the molecular level, the BRAF V600E mutation is present in almost all patients with HCL, providing an important diagnostic clue.
Overall, differential diagnosis of B-CLPDs should integrate multiple dimensions, including morphology, immunophenotype, cytogenetics, and molecular genetics. If a definitive diagnosis still cannot be established after these investigations, lymph node biopsy is recommended for patients with lymphadenopathy to facilitate diagnosis. This represents the overall diagnostic approach to these disorders.
《Oncology Frontier – Hematology Frontier》: With the widespread adoption of flow cytometry, next-generation sequencing (NGS), and FISH, the differential diagnosis of B-CLPDs has entered an era of precision medicine. Could you discuss the value of these technologies in classifying challenging LPD cases? What diagnostic bottlenecks remain in clinical practice, and how can the diagnostic workflow for LPDs be further standardized to improve classification accuracy?
Professor Wei Xu: Morphological assessment forms the foundation of the differential diagnosis of B-CLPDs. For example, HCL is characterized by cells with hair-like cytoplasmic projections, while CLL commonly presents with characteristic smudge cells on peripheral blood smears. However, the fundamental basis for differential diagnosis remains immunophenotypic analysis, because different subtypes possess distinct immunophenotypic markers.
When morphology and immunophenotyping remain insufficient to establish a definitive diagnosis, genetic testing should be incorporated. At the cytogenetic level, conventional karyotyping or FISH can be used to detect specific chromosomal translocations, such as t(14;18) in FL and t(11;14) in MCL, which provide important evidence for disease classification.
If these investigations remain inconclusive, mutation analysis using technologies such as next-generation sequencing (NGS) can provide critical molecular diagnostic information. For example, the BRAF V600E mutation is highly informative for the diagnosis of HCL.
In clinical practice, differential diagnosis of SMZL is particularly challenging. Although splenectomy with pathological examination is considered the diagnostic gold standard, its invasive nature means that clinicians often seek non-invasive minimum diagnostic criteria. For patients who meet these minimum criteria but remain difficult to diagnose, NGS can provide important additional evidence. Detection of mutations in genes such as NOTCH2 and KLF2 can effectively support the diagnosis and differential diagnosis of SMZL.
Expert Profile
Professor Wei Xu
First Affiliated Hospital of Nanjing Medical University (Jiangsu Province Hospital)
Second-level Professor, Chief Physician, Doctoral Supervisor, and Postdoctoral Co-supervisor
Deputy Chair, Hematologic Oncology Committee, Chinese Anti-Cancer Association
Deputy Chair, Lymphoma Committee, China Association of Gerontology and Geriatrics
Deputy Chair, Lymphoma Committee, China Medical Education Association
Standing Committee Member, Lymphoma Committee, Chinese Society of Clinical Oncology (CSCO)
Standing Committee Member, Hematology Committee, Chinese Women’s Physicians Association
Deputy Chair, Hematology Branch, Jiangsu Medical Association
Vice President, Hematology Physicians Branch, Jiangsu Medical Doctor Association
Chair, Lymphoma Committee, Jiangsu Research Hospitals Association
Chair, Jiangsu Anti-Lymphoma Alliance
Chair, Nanjing Hematology Society
