Knoop C, Andrien M, Defleur V, Antonine M, de Francquen P, Goldman M, Estenne M
Department of Chest Medicine, Erasme University Hospital, Brussels, Belgium.
Transplantation. 1994 Dec 27;58(12):1335-8.
Migration of donor cells from the graft to various tissues of the recipient has been demonstrated after different types of solid organ transplants. Currently, the detection of donor cells in the recipient's tissues is most simply performed by polymerase chain reaction (PCR) amplification of a donor-specific gene. In the present study, we first determined in vitro the sensitivity of standard and nested PCR amplification with sequence-specific primers (PCR-SSP) of a donor-specific allele of the HLA-DRB1 gene and then used this technique to assess prospectively blood chimerism in two single-lung (SLT) and one heart-lung (HLT) transplant recipients. Standard PCR-SSP consisted in a single amplification round with sequence-specific primers for the donor-specific DRB1 allele. Nested PCR-SSP consisted in a first round of generic amplification of exon 2 of the DRB1 gene, followed by a second amplification round with primers specific for the donor allele. In vitro, nested PCR-SSP of the donor-specific allele was 1000-fold more sensitive than standard PCR-SSP and allowed the detection of 1 donor cell in 10(5) recipient cells. In vivo, standard PCR-SSP detected donor cells among the recipients' peripheral blood mononuclear cells (PBMCs) only during the first postoperative days, whereas nested PCR-SSP demonstrated their presence until the end of the first postoperative month in patients 1 and 2 and until 3 months after transplantation in patient 3. We conclude that donor cells can be detected in the peripheral blood of SLT and HLT recipients during the first postoperative months and that nested PCR-SSP amplification of a donor-specific HLA-DRB1 allele is much more sensitive than standard PCR-SSP to demonstrate such chimerism.
在不同类型的实体器官移植后，已证实供体细胞可从移植物迁移至受体的各种组织。目前，检测受体组织中的供体细胞最简单的方法是通过聚合酶链反应（PCR）扩增供体特异性基因。在本研究中，我们首先在体外确定了使用序列特异性引物（PCR-SSP）对HLA-DRB1基因的供体特异性等位基因进行标准PCR扩增和巢式PCR扩增的灵敏度，然后使用该技术前瞻性地评估了两名单肺移植（SLT）受者和一名心肺移植（HLT）受者的血液嵌合情况。标准PCR-SSP包括使用针对供体特异性DRB1等位基因的序列特异性引物进行一轮扩增。巢式PCR-SSP包括第一轮对DRB1基因外显子2进行通用扩增，随后第二轮使用针对供体等位基因的特异性引物进行扩增。在体外，供体特异性等位基因的巢式PCR-SSP比标准PCR-SSP敏感1000倍，能够检测出10⁵个受体细胞中的1个供体细胞。在体内，标准PCR-SSP仅在术后最初几天在受体的外周血单核细胞（PBMC）中检测到供体细胞，而巢式PCR-SSP在患者1和患者2术后第一个月末均证实供体细胞存在，在患者3中则在移植后3个月仍能检测到。我们得出结论，在术后最初几个月内可在SLT和HLT受者的外周血中检测到供体细胞，并且扩增供体特异性HLA-DRB1等位基因的巢式PCR-SSP比标准PCR-SSP在证明这种嵌合现象方面更为敏感。
