背景:公共脐带血库(CBBs)在释放之前需要测量脐带血单位(CBU)的效力,允许识别可能不适合造血移植的单位。我们已经开发了一种快速流式细胞术测定,其基于对响应于IL-3刺激的CD34+干细胞的STAT-5磷酸化的测量。
方法:为了使分析从研究环境适应其在CBB监管操作中的实施,我们进行了完整的方法验证以及IL-3-pSTAT5测定结果与集落形成单位测定(CFU)结果的相关性比较.通过流式细胞术分析冷冻保存在小瓶中的总共60个CBUs,以确定灵敏度。特异性,测定内精度,鲁棒性,再现性,和实验室间的测定协议。出于比较目的,还对相同样品进行CFU测定。
结果:测定阈值建立在50%CD34+CD45+pSTAT5+,它提供了100%的灵敏度和98.3%的特异性。测定的平均测定内CV为7.3%。所有结果均符合我们关于用户间和实验室间协议的定性结果接受标准,IL-3刺激时间,解冻后孵育延迟和染色时间。IL-3-pSTAT5测定结果与使用CFU测定测定的总CFU良好相关(r2=0.82,n=56)。
结论:这项研究表明,我们的快速流式细胞术测定法可以成功验证,并且获得的效价数据显示出良好的灵敏度,特异性和鲁棒性。这些结果证明了在CBB操作中实施该测定的可行性,作为验证的效力测定。
BACKGROUND: 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 haematopoietic 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.
METHODS: To adapt the assay from a research setting to its implementation within our CBB regulated operations, we proceded 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 analysed 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.
RESULTS: 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 (r2 = 0.82, n = 56).
CONCLUSIONS: 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 potency assay.