Socié G, Gluckman E, Carosella E, Brossard Y, Lafon C, Brison O
Unité de biologie des cellules souches, LIRB/CEA-DSV and LBMO, Hôpital Saint-Louis, Paris, France.
Blood. 1994 Jan 15;83(2):340-4.
Since our first report in 1989, 26 transplants by means of umbilical cord blood have been reported. Furthermore, systematic studies of the feasibility of using banked placental blood for bone marrow reconstitution of unrelated recipients on a large scale are in progress worldwide. However, already by 1989, it was pointed out that the use of cord blood might be hampered by contamination of neonatal blood with maternal cells contributing unacceptably to graft-versus-host disease (GVHD). In the present study, we used the polymerase chain reaction (PCR) amplification of 2 minisatellite sequences (33.6 and MS 51) to address this question. The sensitivity of PCR amplification of minisatellite sequences is known to be of 1% to 0.1%. This sensitivity has been confirmed in the present study, in which a dilution analysis was performed for each experiment in which cell separation was performed. The inclusion of the dilution experiment in these analyses allowed us to estimate the relative amount of contaminating maternal cells, if any. Among 47 cases (31 whole blood analyses, 10 gradient separations, and 6 subpopulation separations), the coamplification of the 2 minisatellites sequences allowed the discrimination of maternal and neonate alleles in 42 cases (89%). In 1 case, we were able to detect a child-specific allele in a mother's whole blood sample, thus validating our approach to search for maternal cells in cord blood. In a single other case, we were able to detect a maternal-specific allele in the cord blood sample. This maternal specific allele was detected in the whole blood, polymorphonuclear cell, and lymphocyte fractions. Comparison of the signal intensity obtained with these 3 cord blood samples to the result of the dilution experiment performed in the same analysis led to an estimate of 1 to 5% maternal cells in the polymorphonuclear cell fraction and 0.1% to 1% maternal cells in the whole blood and lymphocyte cell fractions. In conclusion, our study indicates that maternal cells are very rarely present in the cord blood collected at birth because we detected them in only 1 of 47 cases. More importantly, when detected, they were present at very low level in the lymphocyte cell fraction (0.1% to 1%). However, although small, this amount of cells may result in GVHD in a susceptible recipient. Because the method we used allows the detection of maternal cells within cord blood from 10(4) nucleated cells, it would thus be of interest in a cord blood banking perspective.
自我们1989年首次报告以来，已有26例通过脐带血进行的移植被报道。此外，全球范围内正在对使用储存的胎盘血大规模重建无关受体骨髓的可行性进行系统研究。然而，早在1989年就有人指出，脐带血的使用可能会因新生儿血液被母体细胞污染而受到阻碍，这些母体细胞对移植物抗宿主病（GVHD）的影响令人无法接受。在本研究中，我们使用聚合酶链反应（PCR）扩增2个微卫星序列（33.6和MS 51）来解决这个问题。已知微卫星序列PCR扩增的灵敏度为1%至0.1%。在本研究中这一灵敏度得到了证实，其中对每个进行细胞分离的实验都进行了稀释分析。在这些分析中纳入稀释实验使我们能够估计污染的母体细胞的相对数量（如果有的话）。在47例（31例全血分析、10例梯度分离和6例亚群分离）中，2个微卫星序列的共扩增在42例（89%）中能够区分母体和新生儿等位基因。在1例中，我们能够在母亲的全血样本中检测到一个儿童特异性等位基因，从而验证了我们在脐带血中寻找母体细胞的方法。在另外仅1例中，我们能够在脐带血样本中检测到一个母体特异性等位基因。在全血、多形核细胞和淋巴细胞组分中都检测到了这个母体特异性等位基因。将这3个脐带血样本获得的信号强度与同一分析中进行的稀释实验结果进行比较，得出多形核细胞组分中母体细胞估计为1%至5%，全血和淋巴细胞组分中母体细胞为0.1%至1%。总之，我们的研究表明，出生时采集的脐带血中母体细胞非常罕见，因为我们在47例中仅在1例中检测到它们。更重要的是，当检测到时，它们在淋巴细胞组分中的含量非常低（0.1%至1%）。然而，尽管数量很少，但这一数量的细胞可能会在易感受体中导致GVHD。因为我们使用的方法能够从10^4个有核细胞中检测脐带血中的母体细胞，所以从脐带血库的角度来看这将是有意义的。
