Flow Cytometric Quantitation of CD34+ Progenitors in Peripheral Blood: An Underutilized Tool in Diagnostic Evaluation of Pancytopenia

Aplastic anemia (AA) is a type of immune-mediated bone marrow (BM) failure characterized by peripheral pancytopenia and marrow hypocellularity. The disease demonstrates decreased to absent CD34+ progenitors, including hematopoietic stem cells (HSCs), due to their diminished production. In normal healthy individuals, small numbers of CD34+ progenitors are detectable in peripheral blood (PB) by flow cytometry. In patients with pancytopenia due to AA, other BM failure syndromes (genetic etiologies) and/or transient bone marrow suppression (viral or other reactive etiologies), PB CD34+ progenitors (CD34+) are expected to be decreased. Conversely, the PB CD34+ population size is expected to be maintained or increased in peripheral causes of cytopenias (destruction and/or sequestration, most immune- or infectious cases). BM CD34+ have been previously well characterized in AA (e.g. Matsui et al, Leukemia 2006), but the features - and associated clinical utility - of PB CD34+ progenitors in routine clinical flow cytometry for evaluation of pancytopenia has not been systematically studied. We hypothesized that review of a large dataset of such cases would inform evidence-based incorporation of PB CD34+ in routine clinical flow cytometry reporting. This study utilized an archival cohort of patients from Children's Hospital Los Angeles of previously healthy patients presenting between Jan 2022 and Dec 2023 with cytopenias of two or three lineages (bi- or pancytopenia) on whom a PB flow cytometry screening panel was performed. The CHLA screening panel utilizes in part a two-tube progenitor- and myeloid-focused antigen combination (M1 and M2 in Wood, Curr Protoc Cytom 2020). The flow cytometry (fcs) files were re-examined to quantify the circulating CD34+CD38+ cell population (CD34+), if present, in each of the two tubes. The average of the two tubes was expressed as a percent (%) of the average of total white blood cells (WBCs). The %CD34+ were correlated with associated clinical and laboratory characteristics including presenting symptoms, complete blood counts, findings on subsequent bone marrow biopsy if performed, and ultimate clinical diagnosis. Of the total study cohort (n=155), ultimate diagnoses to which the cytopenias were ascribed included AA (n=15, 9.7%), infection (n=44, 28.4%), other immune-mediated causes (n=41, 26.5%), metabolic disorders/nutritional deficiencies (n=10, 6.5%) non-hematologic malignancies (n=2, 1.4%) and unknown or non-specific presumed reactive causes (n=40, 25.8%). %CD34+ ranged from 0% to 1.5% (median 0.013%) across the cohort. Among the 18 patients with %CD34+ ≤0.01%, 13 had AA; the other 5 had Factor XII deficiency, infectious mononucleosis, acute pericarditis, and two consistent with leukemoid reactions but unknown diagnoses. The two AA patients with %CD34+ >0.01% had AA secondary to systemic lupus erythematosus and acute hepatitis, respectively. Compared to all non-AA patients, AA patients had significantly lower absolute WBC counts (p<0.0001) and absolute neutrophil counts (p<0.0001). %CD34+ was significantly lower among AA patients (median 0.0055%, range 0-0.045%) compared to all other patients (n=140, median 0.069%, range 0-1.5%, p=0.0076), patients with infection (median 0.045%, range 0.0038-0.62%; p=0.004) and with other immune-mediated processes (median 0.045%, range 0.0085-0.11%; p<0.0001). These results confirm that circulating CD34+ progenitors are significantly decreased in the PB of patients with AA compared to patients with cytopenias of other causes. Conversely, peripheral destruction/sequestration of WBCs as the cause of cytopenias (most cases of systemic inflammatory processes) is associated with increased circulating CD34+ progenitors due to appropriate bone marrow response. This suggests that detection of an expanded circulating CD34+ progenitor population could be a useful piece of clinical data to support a diagnosis of a self-limited immune-mediated process and in turn avoid or postpone more invasive testing such as a bone marrow biopsy. Future directions of this study include quantitation of CD34+ progenitors in subsequently obtained bone marrow aspirate material in this cohort, quantitation of the circulating HSC population (CD34+, CD38dim+) among CD34+ progenitors, and establishment of a normal range of circulating CD34+ progenitors in normal healthy individuals.