Girard Mélissa, Laforce-Lavoie Audrey, de Grandmont Marie Joëlle, Cayer Marie-Pierre, Fournier Diane, Delage Gilles, Thibault Louis
Research and Development, Héma-Québec, Québec, Québec, Canada.
Public Cord Blood Bank.
Transfusion. 2017 Aug;57(8):1956-1967. doi: 10.1111/trf.14147. Epub 2017 May 4.
Different methods are used by cord blood banks to prepare samples for sterility testing. Suboptimal methods can result in the release of contaminated products. In our organization, samples are prepared by diluting the final product in RPMI-1640 medium. In this work, we have compared our method with different approaches to verify whether optimization should be sought.
Cord blood units (n = 6 units per bacterial strain) characterized to contain inhibitory substances or not were inoculated (10 colony-forming units/mL) with Streptococcus agalactiae, Staphylococcus epidermidis, Klebsiella pneumoniae, Escherichia coli, or Bacteroides fragilis. After plasma and red blood cell removal, stem cell concentrates were diluted in RPMI-1640, thioglycollate, or the unit's plasma. These products, as well as final product, plasma, and red blood cell fractions, were held from 0 to 72 hours at 20 to 24°C before inoculation in culture bottles and detection using the BacT/ALERT 3D system.
Dilution of cell concentrates in RPMI-1640 allowed bacterial detection in 93.3% of noninhibitory cord blood samples after a 24-hour storage period. Thioglycollate medium better promoted bacterial growth in inhibitory cord blood samples that were held for 72 hours before testing (66.7%) compared with RPMI-1640 (45.0%). Less than 33% of all spiked plasma samples were detected by the BacT/ALERT 3D system.
Diluting cord blood samples in culture medium containing bacterial growth promoting substances is a suitable option for sterility testing, whereas the use of plasma should be proscribed, because it might lead to false-negative results. Because inhibitory substances affect bacterial growth, inoculation of culture bottles should be done rapidly after sample preparation.
不同的脐带血库采用不同的方法来制备用于无菌检测的样本。方法欠佳可能导致放行受污染的产品。在我们机构，样本是通过将最终产品在RPMI-1640培养基中稀释来制备的。在本研究中，我们将我们的方法与不同方法进行了比较，以验证是否应寻求优化。
对特征为含有或不含有抑制物质的脐带血单位（每种细菌菌株6个单位）接种无乳链球菌、表皮葡萄球菌、肺炎克雷伯菌、大肠埃希菌或脆弱拟杆菌（10个菌落形成单位/毫升）。去除血浆和红细胞后，将干细胞浓缩物在RPMI-1640、硫乙醇酸盐或该单位的血浆中稀释。这些产品以及最终产品、血浆和红细胞部分在接种到培养瓶中并使用BacT/ALERT 3D系统检测之前，于20至24°C下保存0至72小时。
在RPMI-1640中稀释细胞浓缩物，在24小时储存期后，93.3%的无抑制作用的脐带血样本中可检测到细菌。与RPMI-（45.0%）相比，硫乙醇酸盐培养基能更好地促进在检测前保存72小时的有抑制作用的脐带血样本中的细菌生长（66.7%）。BacT/ALERT 3D系统检测到的所有加标血浆样本不到33%。
在含有促进细菌生长物质的培养基中稀释脐带血样本是无菌检测的合适选择，而应禁止使用血浆，因为它可能导致假阴性结果。由于抑制物质会影响细菌生长，样本制备后应尽快接种培养瓶。
