Background:

Inherited bone marrow failure syndromes (IBMFS), such as Fanconi anemia, and acquired aplastic anemia (AAA) both present with bone marrow hypoplasia and pancytopenia. IBMFS arises from germline mutations affecting key cellular processes, including telomere maintenance, DNA repair pathways, and ribosomal biogenesis. In contrast, AAA is an immune-mediated disorder characterized by autoreactive, oligoclonally expanded T cells targeting hematopoietic progenitor cells. Comprehensive genetic testing, including next-generation sequencing (NGS), has become a standard approach for evaluating the etiology of bone marrow failure. However, timely differentiation between IBMFS and AAA remains clinically challenging, as NGS results may be delayed or inconclusive despite exhaustive testing.

Given the importance of distinguishing IBMFS from AAA, particularly to guide decisions regarding hematopoietic stem cell transplantation (HSCT) versus immunosuppressive therapy (IST), we evaluated the spectrum and size of oligoclonally expanded T cell subsets in bone marrow samples using highly sensitive multicolor flow cytometry. We specifically employed an anti–T-cell receptor β-chain constant region 1 (TRBC1) antibody to assess T cell clonality and investigated its potential utility in differentiating AAA from IBMFS.

Methods:

Bone marrow samples from 239 subjects (median age: 28 years; interquartile range [IQR]: 16–45 years; 126 males [52.7%], 113 females [47.3%]) were analyzed using a 10-color flow cytometry panel. The cohort included 167 patients with AAA [negative for germline mutations by whole exome sequencing], 18 genetically confirmed IBMFS cases, and 54 controls [uninvolved bone marrow samples of patients with non-Hodgkin lymphoma]. After excluding γδ T cells, detailed immunophenotyping of CD4pos and CD8pos T cell compartments—specifically naive, central memory, effector memory, and terminal effector subsets—was performed. Each subset was further stratified based on CD57 expression, and clonality was assessed in 16 defined subsets using TRBC1 expression.

Clonality was defined as TRBC1 expression in ≥90% or ≤10% of a given subset, with a minimum of 50 events required. We compared the frequency and size of T cell clones across diagnostic groups and examined their associations with age, disease severity, and telomere length.

Results:

T cell subset distributions were similar between AAA and IBMFS groups after adjusting for age. However, clonal expansion patterns differed significantly. Clonal T cell expansions were identified in 32.6% of patients, predominantly in CD57pos effector and terminal effector subsets. The most frequent clonal expansions were observed in CD4pos effector (35%) and CD8pos terminal effector (31%) compartments. Clone frequency increased with age and correlated with shorter telomere length, particularly among CD8+ clones (p = 0.049).

Importantly, clone sizes were significantly larger in AAA compared to IBMFS. CD8pos T cell clones comprising >5% of total T cells demonstrated 86% sensitivity and 96% positive predictive value (PPV) for distinguishing AAA from IBMFS. A clone size >10% in either CD4pos or CD8pos compartments conferred 100% specificity for an AAA diagnosis.

Conclusion:

Flow cytometric assessment of T cell clonality, particularly clone size, provides a valuable adjunct to genetic testing for differentiating AAA from IBMFS. While overall T cell subset distributions were comparable, the presence of large CD8pos T cell clones (>5%) was a robust discriminator favoring AAA, with high sensitivity and PPV. These expanded clones were predominantly CD57pos effector cells and demonstrated strong associations with shortened telomere length, suggesting unique immunopathogenic mechanisms in AAA. Thus, TRBC1-based clonality analysis via flow cytometry offers a rapid and informative tool to support clinical decision-making in bone marrow failure syndromes.

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2025
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