脐血采分和长期冻存后细菌培养的研究

Study on bacteria contamination during the collection, processing and storage of umbilical cord blood

目的探讨脐血采分储存中细菌污染的种类、途径和规律性以及深低温冻存对细菌活力的影响。方法在2000年至2007年脐血干细胞采分常规工作中，留取有核细胞分离后血浆-红细胞悬液10ml，分别注入需氧和厌氧细菌培养瓶，用BacT／ALERT3D-480全自动血液培养系统培养7d，细菌培养阳性者作废弃处理。同时选取细菌阳性的新鲜脐血87份进一步培养24h，获得纯培养后，将革兰阳（阴）性菌鉴定卡放入全自动微生物分析系统读数孵育器，分别进行需氧菌与厌氧菌鉴定。另外，为观察深低温冻存对细菌的影响，取出冻存6-7年的细菌阳性脐血96份，37℃速融后，取有核细胞浓缩物10ml，用上述方法做二次细菌培养检测。结果2000年至2007年采集脐血19062份，细菌培养阳性336份，细菌污染率为1．8％；作细菌鉴定8I份，发现兼性生长58份（67％），专性需氧生长38份（43．7％），专性厌氧生长17份（19．5％）；革兰阴性菌占68％，革兰阳性菌占32％。细菌种类：大肠埃希菌最常见，占25．3％，其次为中间链球菌占14．9％，紫色色杆菌占9．2％。96份细菌鉴测阳性并移至液氮保存6-7年的脐血标本中，经复检仍有83份（86％）保持细菌活性。结论2000年至2007年脐血采分过程中细菌污染率为1．8％，污染菌在液氮冻存6-7年后86％细菌仍存活。故在产房采集脐血时应加强无菌措施，在临床使用冻存于细胞时应加强细菌复检。

Objective To evaluate bacteria contamination during collection, processing and storage of cord blood to gain insight into contamination mechanism and direct prevention. Methods Fresh cord blood was separated by hydroxyethyl starch （ HES ） to harvest nucleated cells. The bacteria contamination was tested by culturing 10 ml plasma-red cells with BacT/ALERT 3D-480 automatic blood culture system. Total 87 positive samples were further identified for bacteria species. Ninety six cord blood nucleated cells concentrate with bacte- ria positive stored in liquid nitrogen（LN2） for 6-7 years were thawed at 37 ℃ and re-cultured for bacteria analy- sis. Results We collected 19 062 umbilical cord blood. Among them, 336 was bacteria positive （ contamination rate 1.8 % ）. Eighty-seven positive samples were further investigated with facultative bacteria 58 （66.7%）, aero- bic 38（43. 7% ） and anaerobic 17（ 19. 5% ） ,Gram- negative accounted for 68% while positive 32%. The most frequent bacteria were Escherichia coli （ 25.3% ）, Streptacoccus intermediate （ 14. 9% ） and Chromobacteria violaceum（ 9. 2% ）. Ninety-six nucleated cells concentrate with bacteria positive were cryopreserved at liquid nitrogen for researching. Of them ,83 samples（ 86% ） showed positive of bacteria culture after deep-low tempera- ture storage for 6-7 years. Conclusions Bacteria contamination rate of the cord blood collection,processing and storage in 2000 -2007 was 1.8%. Stored in liquid nitrogen for 6-7 years ,the viability of bacteria was 86%. The aseptic procedures of cord blood collection in delivery room should be intensified. The bacteria re.-culture follow- ing thawing of cord blood cells is necessary before clinical transfusion.