Enumeration of CD34-CD133+ Haematopoietic Progenitor Cells in Cord Blood

Cord blood unit (CBU) selection for allogeneic hematopoietic stem cell transplantation relies heavily on haematopoietic progenitor cell (HPC) enumeration, with CD34+ dose strongly associated with engraftment and survival. CD133 marks more primitive progenitor populations, including rare early-stage CD34-CD133+ HPC that exhibit high haematopoietic activity in vivo but are not routinely assessed due to their scarcity. To determine whether early-stage CD34-CD133+ HPC can be reliably detected and enumerated in fresh precryopreservation cord blood samples using a modified International Society of Hematotherapy and Graft Engineering (ISHAGE) gating strategy, and to evaluate the proportional relationship between early (CD34-CD133+) and more mature (CD34+) progenitor subsets. Exploratory analyses were also performed to assess associations between CD34+ proportion and transplant outcomes. Fresh buffy coat-enriched CB samples from CBUs meeting Therapeutic Goods Administration and Foundation for the Accreditation of Cellular Therapy-NetCord banking thresholds were analyzed using the Whole Blood CD34/CD133 Stem Cell Inspector kit (Miltenyi) on a validated FACSLyric platform. Cryopreserved samples were not included in the study. An ISHAGE-based approach was adapted by incorporating CD133 as a primary positive marker. Forty-one randomly selected CBUs were analyzed for progenitor subset relationships. Retrospective clinical outcomes were analyzed for single-unit transplants released by Sydney Cord Blood Bank (2008 to 2017; n = 152). Following application of predefined eligibility criteria, survival and engraftment analyses were performed in 104 evaluable patients. These patients were then categorized by infused total nucleated cell (TNC) dose (>7 × 10⁷/kg versus 3 to 6.9 × 10⁷/kg) and CD34+ content ≥0.45% versus <0.45%, where 0.45% represented the mean CD34+ proportion observed in the flow cytometry cohort (n = 41). CD34-CD133+ HPCs were detectable in all CB samples (n = 41). CD34+ frequency within the CD45+ white blood cell compartment ranged from 0.08% to 1.88% (n = 41). A strong inverse correlation was observed between the proportion of CD34+ cells and the proportion of CD34- cells within the CD133+ compartment (Spearman r = -0.795, P < .0001), with the relationship best described by a power regression model (R² = 0.64). CD34-CD133+ cells were significantly smaller than CD34+ HPC (P < .0001), consistent with a quiescent phenotype. CD45 expression shifted with maturation, supporting phenotypic staging from CD45^brightCD34-CD133+ (early) to CD45^dimCD34+CD133+ (intermediate) and CD45^brightCD34+CD133- (late). In exploratory outcome analyses, infused TNC dose correlated with infused CD34+ dose (P < .0001, R² = 0.59), whereas CD34+ proportion within TNC did not correlate with TNC dose (P = .07, R² = 0.02). In high TNC grafts, overall survival did not differ by CD34+ proportion. In lower TNC grafts, overall survival was higher when CD34+ proportion was <0.45% (of TNC), while neutrophil engraftment was faster when CD34+ proportion was >0.45% (median 17 versus 24 d; P = .0044), with a trend toward faster platelet engraftment (median 39 versus 49 d; P = .077). A modified ISHAGE strategy incorporating CD133 enables detection and enumeration of rare CD34-CD133+ HPC in fresh precryopreservation cord blood samples. Early and mature progenitor subsets demonstrate an inverse proportional relationship and distinct CD45 maturation profiles. These findings support prospective studies incorporating direct CD133 assessment to determine whether CD34-CD133+ progenitor content is associated with cord blood transplantation outcomes.