Development and validation of a rapid, aldehyde dehydrogenase bright-based cord blood potency assay

Abstract

Banked, unrelated umbilical cord blood provides access to hematopoietic stem cell transplantation for patients lacking matched bone marrow donors, yet 10% to 15% of patients experience graft failure or delayed engraftment. This may be due, at least in part, to inadequate potency of the selected cord blood unit (CBU). CBU potency is typically assessed before cryopreservation, neglecting changes in potency occurring during freezing and thawing. Colony-forming units (CFUs) have been previously shown to predict CBU potency, defined as the ability to engraft in patients by day 42 posttransplant. However, the CFU assay is difficult to standardize and requires 2 weeks to perform. Consequently, we developed a rapid multiparameter flow cytometric CBU potency assay that enumerates cells expressing high levels of the enzyme aldehyde dehydrogenase (ALDH bright [ALDH(br)]), along with viable CD45(+) or CD34(+) cell content. These measurements are made on a segment that was attached to a cryopreserved CBU. We validated the assay with prespecified criteria testing accuracy, specificity, repeatability, intermediate precision, and linearity. We then prospectively examined the correlations among ALDH(br), CD34(+), and CFU content of 3908 segments over a 5-year period. ALDH(br) (r = 0.78; 95% confidence interval [CI], 0.76-0.79), but not CD34(+) (r = 0.25; 95% CI, 0.22-0.28), was strongly correlated with CFU content as well as ALDH(br) content of the CBU. These results suggest that the ALDH(br) segment assay (based on unit characteristics measured before release) is a reliable assessment of potency that allows rapid selection and release of CBUs from the cord blood bank to the transplant center for transplantation.

© 2016 by The American Society of Hematology.

Figures

Figure 1

Flowchart of the ALDH potency assay performed on attached segments of CBUs requested for CT for donor selection. 7-AAD, 7-aminoactinomycin D; FTA, Fast Technology Analysis; GlyA, glycophorin A; HSA/PBS, human serum albumin/phosphate-buffered saline; HPCA, hematopoietic progenitor cell assay.

Figure 2

Potency assay gating. (A) Debris-free gate. (B) Gating of the nonviable (7-AAD+) and RBC (PE-Cy5-GlyA+) cells. (C) Gating of the PE-CD45+ cells. (D) Gating of APC-CD34+ cells. (E) Gating of ALDHbr cells. (F) Gating of cells stained with the Aldecount reagent in the presence of the inhibitor DEAB. BAA, BODIPY aminoacetate; FSC, forward scatter; SSC, side scatter.

Figure 3

Intermediate precision assessment. CV of segments from 5 CBUs (UCB1-UCB5) analyzed over 6 days using 2 operators (Op1 and Op2) and 2 Instruments (In1 and In2). The target specification was a CV ≤20%.

Figure 4

Validation of specificity. Purified ALDHbr cells (20 000) were spiked into varying amounts of CBU cells (1 × 106, 3 × 106, 5 × 106, and 10 × 106). Segments were created and cryopreserved. The segments were subsequently thawed and analyzed with the potency assay, with a target of collecting 5000 ALDHbr cells. The target of 4000 to 6000 cells was met at all concentrations.

Figure 5

Comparison of cellular components of CT segments. (A) ALDHbr in viable CD45+ vs CFUs. (B) CD34+ in viable CD45+ vs CFUs. (C) ALDHbr in viable CD45+ vs CD34+ in viable CD45+. N = 3908.

Figure 6

Comparison of CT segments in fresh or thawed CBUs. (A) Samples taken from fresh CBUs before cryopreservation between July 2007 and August 2009 were assayed for ALDHbr and compared with the results of the CT assay of segments from those units. N = 596. Red line indicates theoretical equality. (B) Research CBUs stored at the CCBB were selected. The bags were thawed with the segments and tested for ALDHbr. N = 60. Red line indicates theoretical equality.

Figure 7

ALDHbr impact on engraftment. Impact of ALDHbr measured on segments during CT on engraftment of the corresponding unit. Probability plots are shown for the units with an ALDHbr >0.5% of viable CD45+ or the units with an ALDHbr <0.5% of viable CD45+ in reaching an absolute neutrophil count of 500/μL. N = 78.