Pujal Josep-Maria, Gallardo David
Translational Research Laboratory, Institut Català d'Oncologia, Hospital Duran i Reynals, Avda Gran Via s/n, Km 2.7, 08907 L'Hospitalet de Llobregat, Barcelona, Spain.
Exp Biol Med (Maywood). 2008 Sep;233(9):1161-70. doi: 10.3181/0802-RM-35. Epub 2008 Jun 5.
Peripheral blood microchimerism after pregnancy or solid organ transplantation has been widely studied, but a consensus on its detection has not yet been adopted. The objective of this study was to establish a panel of reproducible molecular polymerase chain reaction (PCR)-based methods for detection and quantification of foreign cells in an individual. We analyzed length polymorphisms generated by short tandem repeat (STR) and variable number tandem repeat (VNTR) markers. Human leukocyte antigen (HLA)-A and -B polymorphisms were detected by reference strand conformation analysis (RSCA). Class II polymorphisms on HLA-DRB1 locus were analyzed both by classical PCR-sequence-specific primers (SSP) and by quantitative PCR (Q-PCR). Also, sex-determining region-y gene (SRY) gene allowed specific male donor discrimination and quantification by Q-PCR in female recipients. Binomial statistical distribution analysis was used for each molecular technique to determine the number of PCR replicates of each sample. This analysis allowed the detection of the lowest detectable microchimerism level, when present. We could detect microchimerism in more than 96% and more than 86% of cases at levels as low as 1:10(5) and 1:10(6) donor per recipient cells (DPRC), respectively, using Q-PCR for SRY or for nonshared HLA-DRB1 alleles. These techniques allowed as low as 1 genome-equivalent cell detection. Lower levels (nanochimerism) could be detected but not quantified because of technique limitations. However, classical PCR methods allowed detection down to 1:10(4) DPRC for HLA-DRB1 PCR-SSP. The clinical application of these techniques in solid organ transplanted recipients showed microchimerism levels ranging from 1:10(4) to 1:10(6) DPRC after kidney or heart transplantation, and 1 log higher (1:10(3) to 1:10(6) DPRC) after liver transplantation. In conclusion, the standardization of molecular microchimerism detection techniques will allow for comparable interpretation of results in microchimerism detection for diagnostic or research studies.
妊娠或实体器官移植后的外周血微嵌合体已得到广泛研究，但尚未就其检测方法达成共识。本研究的目的是建立一组基于分子聚合酶链反应（PCR）的可重复方法，用于检测和定量个体中的外来细胞。我们分析了短串联重复序列（STR）和可变数目串联重复序列（VNTR）标记产生的长度多态性。通过参考链构象分析（RSCA）检测人类白细胞抗原（HLA）-A和-B多态性。通过经典的PCR序列特异性引物（SSP）和定量PCR（Q-PCR）分析HLA-DRB1基因座上的II类多态性。此外，性别决定区Y基因（SRY）可通过Q-PCR在女性受者中进行特异性男性供体鉴别和定量。对每种分子技术采用二项式统计分布分析来确定每个样本的PCR重复次数。该分析能够检测到最低可检测微嵌合体水平（如果存在）。使用针对SRY或非共享HLA-DRB1等位基因的Q-PCR，我们分别可以在超过96%和超过86%的病例中检测到低至1:10⁵和1:10⁶供体/受体细胞（DPRC）水平的微嵌合体。这些技术能够检测到低至1个基因组等效细胞。由于技术限制，可以检测到更低水平（纳米嵌合体）但无法定量。然而，经典PCR方法对于HLA-DRB1 PCR-SSP可检测到低至1:10⁴ DPRC。这些技术在实体器官移植受者中的临床应用显示，肾或心脏移植后微嵌合体水平为1:10⁴至1:10⁶ DPRC，肝移植后则高1个对数级（1:10³至1:10⁶ DPRC）。总之，分子微嵌合体检测技术的标准化将有助于在诊断或研究中对微嵌合体检测结果进行可比的解读。
