Hernigou P, Marinello G, Dormont D
Service d'Orthopédie, Hôpital Henri Mondor, Créteil.
Rev Chir Orthop Reparatrice Appar Mot. 1998 Oct;84(6):493-500.
The purpose of this study was to determine, when assuming the worst case scenario of bone contamination, the sterility assurance level in bone transplantation treated by irradiation and after donor screening.
The virus: We employed the HIV-1, LAV-1 as our reference strain, which has always been cultured on normal human cells (and has therefore never been in contact with cell lines). To test viral virulence, we used a very sensitive cell line: MT2 cell line, a T lymphoblastoid cell line which is HTLV-I positive and is sensitive to HIV-1/LAV-1 infection. It was maintained in RPMI 1640 culture medium containing 10 per cent heat-inactivated fetal calf serum (Boehringer Mannheim), 2 mM L-glutamine (Boehringer Mannheim) and 1 per cent antibiotics (PSN 100 x, Gibco). Cell concentration was 100,000 cells per milliliter. Irradiation was performed using an accelerator delivering electrons of 6.2 MeV providing several tens of kilograys in a few seconds. The dose delivered was checked by placing alanine dosimeters at the site of the HIV aliquots. Alanine is a solid amino acid which, when irradiated, gives rise to free radicals; these were counted using electron paramagnetic resonance (relative standard deviation of less than 1 per cent).
25 kilograys irradiation of 316,227 TCID50/ml at -80 degrees C led to a considerable titer reduction (3 logs) with 316 TCID50/ml remaining. According to the technique's sensitivity (100 TCID50/ml) and to the sensitivity with which the dose of irradiation can be measured (less than 1 per cent), the D10 value with "99 per cent confidence limits" was between 8.3 kilograys and 7.2 kilograys. Assuming the worst characteristics of irradiation, we have chosen 8.3 kilograys as the D10 value for -80 degrees C.
Considering a mean level of contamination of 30 TCID50 per milliliter of plasma during the asympomatic phase of infection, the reduction of the transmission risk of HIV by a femoral head allograft was calculated in the best case to be only 5 per cent when performed at 25 kilograys. Irradiation cannot be considered as a process able to achieve sterility of the graft and has to be associated to an extensive screening. However irradiation associated with appropriate screening of donors decreases the risk of transmission of the disease under one out of one million in femoral allograft implantation.
本研究的目的是在假设骨污染最坏情况的前提下，确定经辐照及供体筛查后骨移植中的无菌保证水平。
病毒：我们采用HIV-1、LAV-1作为参考毒株，该毒株一直培养于正常人细胞（因此从未接触过细胞系）。为检测病毒毒力，我们使用了一种非常敏感的细胞系：MT2细胞系，一种T淋巴母细胞系，其为HTLV-I阳性且对HIV-1/LAV-1感染敏感。它保存在含有10%热灭活胎牛血清（勃林格殷格翰）、2 mM L-谷氨酰胺（勃林格殷格翰）和1%抗生素（PSN 100x，Gibco）的RPMI 1640培养基中。细胞浓度为每毫升100,000个细胞。使用一台加速器进行辐照，该加速器提供6.2 MeV的电子，在几秒内可提供几十千戈瑞的剂量。通过在HIV等分试样部位放置丙氨酸剂量计来检查所输送的剂量。丙氨酸是一种固体氨基酸，辐照时会产生自由基；使用电子顺磁共振对这些自由基进行计数（相对标准偏差小于1%）。
在-80℃下对316,227 TCID50/ml进行25千戈瑞的辐照导致滴度大幅降低（3个对数级），剩余316 TCID50/ml。根据该技术的灵敏度（100 TCID50/ml）以及辐照剂量的测量灵敏度（小于1%），“99%置信限”下的D10值在8.3千戈瑞至7.2千戈瑞之间。假设辐照的最坏特性，我们选择8.3千戈瑞作为-80℃下的D10值。
考虑到感染无症状期血浆中每毫升平均污染水平为30 TCID量单位，在25千戈瑞下进行时，股骨头同种异体移植降低HIV传播风险的最佳情况下仅为5%。辐照不能被视为一种能够实现移植物无菌的过程，必须与广泛的筛查相结合。然而，与对供体进行适当筛查相关的辐照可将股骨同种异体移植植入中疾病传播风险降低至百万分之一以下。
