Ramirez-Arcos Sandra, Kou Yuntong, Yang Lin, Perkins Heather, Taha Mariam, Halpenny Mike, Elmoazzen Heidi
Product and Process Development, Canadian Blood Services Centre for Innovation.
National Public Cord Blood Bank, Canadian Blood Services, Ottawa, Ontario, Canada.
Transfusion. 2015 Aug;55(8):1985-92. doi: 10.1111/trf.13050. Epub 2015 Mar 11.
Sterility testing for cord blood (CB) products is mandatory to prevent transplantation-transmitted microbial infections. Here, the automated BacT/ALERT (bioMérieux) culture system was validated to detect microbial contamination in CB units processed at the Canadian National Public Cord Blood Bank.
A three-phase validation was developed. CB units were prepared with pentastarch (Phases 1 and 2) or hetastarch (Phase 3). In Phase 1, CB was spiked with approximately 100 colony-forming units/mL of Pseudomonas aeruginosa, Klebsiella pneumoniae, Staphylococcus aureus, Staphylococcus epidermidis, Bacteroides fragilis, and Candida albicans. Plasma (8 mL) and buffy coat (BC; 0.5 and 8 mL) were inoculated into culture bottles. In Phases 2 and 3, a mix of red blood cells (RBCs) and plasma (4 mL each) was used as the inoculant. In Phase 3, Aspergillus brasiliensis was added as a test organism and microbial concentrations in the by-product RBCs and plasma were determined. The BC fractions were cryopreserved and tested 3 months later.
In Phase 1, bacteria failed to grow in CB units containing antibiotics. Thus, antibiotic-free units were used for the other phases. C. albicans was not always captured in plasma, but using a mix of RBCs and plasma, all organisms were detected. The use of pentastarch or hetastarch did not affect microbial recovery. C. albicans and A. brasiliensis were preferentially recovered in RBCs and BC. Cryopreservation did not affect microbial survival during CB processing.
A mix of plasma and RBCs is appropriate for CB sterility testing. Interestingly, fungi preferentially segregate to cellular fractions. The clinical significance of the bactericidal /or bacteriostatic effect of antibiotics in CB merits further investigation.
脐血（CB）产品的无菌检测对于预防移植传播的微生物感染至关重要。在此，对自动化BacT/ALERT（生物梅里埃公司）培养系统进行了验证，以检测在加拿大国家公共脐血库处理的CB单位中的微生物污染情况。
开展了一个分为三个阶段的验证过程。CB单位使用喷他淀粉（第1和第2阶段）或羟乙基淀粉（第3阶段）进行制备。在第1阶段，向CB中加入约100菌落形成单位/毫升的铜绿假单胞菌、肺炎克雷伯菌、金黄色葡萄球菌、表皮葡萄球菌、脆弱拟杆菌和白色念珠菌。将血浆（8毫升）和 Buffy 层（BC；0.5毫升和8毫升）接种到培养瓶中。在第2和第3阶段，使用红细胞（RBC）和血浆的混合物（各4毫升）作为接种物。在第3阶段，添加巴西曲霉作为测试微生物，并测定副产品RBC和血浆中的微生物浓度。BC部分进行冷冻保存，并在3个月后进行检测。
在第1阶段，含有抗生素的CB单位中细菌未能生长。因此，其他阶段使用不含抗生素的单位。白色念珠菌并非总能在血浆中被检测到，但使用RBC和血浆的混合物时，所有微生物均能被检测到。喷他淀粉或羟乙基淀粉的使用不影响微生物的回收率。白色念珠菌和巴西曲霉在RBC和BC中优先被回收。冷冻保存不影响CB处理过程中微生物的存活情况。
血浆和RBC的混合物适用于CB无菌检测。有趣的是，真菌优先分离到细胞部分。CB中抗生素的杀菌/或抑菌作用的临床意义值得进一步研究。
