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

Abstract

• Cord blood content of ALDH br cells correlates well with CFUs and may act as a surrogate potency assay for cord blood units.• ALDH br cells in segments are assayed rapidly, allowing potency results to be used for release of the unit from a public cord blood bank.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 dehydroge- 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.

The aldehyde dehydrogenase cord blood potency assay excludes early apoptotic cells

“…16 Also, as has been shown by Duggleby and colleagues, measurement of viable CD341 (CD341 7-AAD2) cells includes substantial fractions of early apoptotic cells (CD341 7-AAD2 annexin V1), and exclusion of these in measurements improves conformity with the functional CFU assay. 7 The results from our study together with previously published data, 6,10 suggest that the ALDH assay might serve well as a surrogate for the CFU assay, both regarding the measurement of the number of HSCs in a particular CBU and also to appreciate the functionality of the cells pretransplant. Hence, we found no nonviable and very few apoptotic ALDH1 cells compared to the CD341 cell fraction, indicating that very few ALDH1 cells compared to CD341 cells were nonfunctional.…”

“…9 Cells high in ALDH activity (ALDH1 cells) correlate better than viable CD341 cells with CFU-GM in frozen thawed CBUs, according to a recent study by Shoulars and coworkers. 10 We hypothesized that the excellent correlation between CFU-GM and ALDH1 cells in frozen-thawed CB as presented by Duggleby and colleagues 7 might reflect that the ALDH assay excludes early apoptotic cells. To investigate this, we designed a protocol for simultaneous staining of viable and apoptotic CD341 and ALDH1 cells using 7-AAD and annexin V in frozen-thawed CBUs.…”

“…An alternative, promising approach is the aldehyde dehydrogenase (ALDH) assay (Aldeflour, Stemcell Technologies) building on increased enzyme activity in HSCs . Cells high in ALDH activity (ALDH+ cells) correlate better than viable CD34+ cells with CFU‐GM in frozen thawed CBUs, according to a recent study by Shoulars and coworkers …”

Post‐thaw viability of cryopreserved peripheral blood stem cells (PBSC) does not guarantee functional activity: important implications for quality assurance of stem cell transplant programmes

“…In contrast, the standardization of flow cytometric CD34 + cell counting between transplantation centres has been remarkably successful (Barnett et al, 1998;Gratama et al, 2003) and may present a model for a flow based test where viability relies not on simple permeability but on metabolic function, such as aldehyde dehydrogenase (Shoulars et al, 2016) and Syto16 activity. This standard could be met by sessional colony assays for HPC function however, as previously noted, standardization issues remain a barrier for widespread adoption.…”

Optimal Practices in Unrelated Donor Cord Blood Transplantation for Hematologic Malignancies

“…As a result of center experience and such data, all of our 6 centers consider the Bank of origin in unit selection. If unit segment potency testing can be standardized 44 and are shown to reflect unit content, such assays may assist in pre- and post-thaw quality assessment. In the interim, post-thaw assays of HSC potency 43 are critical as they are available within hours of thaw at the transplant center making the shipment of a back-up unit possible in the unlikely event of a poor quality unit 45 .…”