Validation of a rapid potency assay for cord blood stem cells using phospho flow cytometry: the IL-3-pSTAT5 assay

Abstract

Public cord blood banks (CBBs) are required to measure cord blood units (CBUs) potency before their release, allowing for the identification of units that may be unsuitable for hematopoietic transplantation. We have developed a rapid flow cytometry assay based on the measurement of STAT-5 phosphorylation of CD34+ stem cells in response to IL-3 stimulation: the IL-3-pSTAT5 assay. To adapt the assay from a research setting to its implementation within our CBB regulated operations, we proceeded with a full method validation and a correlation comparison of the IL-3-pSTAT5 assay results with the colony-forming unit assay (CFU) results. A total of 60 CBUs cryopreserved in vials were analyzed by flow cytometry to determine the sensitivity, specificity, intra-assay precision, robustness, reproducibility and inter-laboratory agreement of the assay. The CFU assay was also done on the same samples for comparison purposes. The assay threshold was established at 50% CD34+CD45+pSTAT5+, which provides a 100% sensitivity and a 98.3% specificity. An average intra-assay CV of 7.3% was determined. All results met our qualitative results acceptance criteria regarding the inter-user and inter-laboratory agreements, IL-3 stimulation time, post-thaw incubation delay and staining time. The IL-3-pSTAT5 assay results correlated well with the total CFU determined using the CFU assay (r squared = 0.82, n = 56). This study shows that our rapid flow cytometry assay can be successfully validated and that the potency data obtained display good sensitivity, specificity and robustness. These results demonstrate the feasibility of implementing this assay within CBB operations, as a validated CB potency assay.

Multicenter evaluation of the IL‐3‐pSTAT5 assay to assess the potency of cryopreserved stem cells from cord blood units: The BEST Collaborative study

“…The study used an acceptance threshold of ≥50.0% for CD34+/CD45+/pSTAT5+ cells, which closely corresponded to the Youden's index from a ROC curve established during our internal validation. 11 One of the goals of this study was to validate this threshold in a multicenter setting. To this end, a ROC curve was determined using the data from the study (Figure 1D).…”

“…The two other samples were similarly processed but were exposed to a transient warming event of 2 hours at −20°C (“abnormal samples”) and were thus expected to exhibit <50% of CD34+/CD45+/pSTAT5+ cells after IL‐3 stimulation. The 50% acceptance threshold maximizes the sum of the assay's sensitivity and specificity (Youden's index), as determined in a previous validation of the method 11 . Each center also received all required reagents, a detailed written protocol, a work sheet to document each step, and a video of the protocol.…”

“…Each center received two "practice samples," which could be used to familiarize themselves with the protocol, and 10 "test samples" for the study. Eight of the ten test samples were CBU samples processed and cryopreserved according to SOPs ("normal samples"); these samples were expected to exhibit ≥50% of CD34+/CD45+/pSTAT5+ cells-the acceptance threshold of the assay (internal validation 11 ). The two other samples were similarly processed but were exposed to a transient warming event of 2 hours at À20°C ("abnormal samples") and were thus expected to exhibit <50% of CD34+/CD45+/pSTAT5+ cells after IL-3 stimulation.…”

“…The 50% acceptance threshold maximizes the sum of the assay's sensitivity and specificity (Youden's index), as determined in a previous validation of the method. 11 Each center also received all required reagents, a detailed written protocol, a work sheet to document each step, and a video of the protocol.…”

“…The assay is based on the phosphorylation of STAT5 in CD34+/CD45+ cells following a short IL‐3 stimulation 10 . This assay has been validated at Héma‐Québec's CBB 11 ; however, multiple factors might affect the performance of this assay across laboratories, including experience with flow cytometry, ability to handle CBU samples, and the type of flow cytometer used. Through the Biomedical Excellence for Safer Transfusion (BEST) Collaborative, a multicenter, international study was initiated to assess the inter‐laboratory agreement of the results produced by this assay and to obtain feedback from potential end users.…”